HMGN2 peptides and related molecules that selectively home to tumor blood vessels and tumor cells

ABSTRACT

The present invention provides a conjugate which contains a therapeutic moiety linked to a homing molecule that selectively homes to tumor blood vessels and tumor cells and that specifically binds the receptor bound by peptide KDEPQRRSARLSAKPAPPKPEPKPKKAPAKK (SEQ ID NO: 9). Methods of directing a conjugate of the invention to tumor blood vessels and tumor cells and of using a conjugate to treat cancer also are provided.

This application claims benefit of the filing date of U.S. ProvisionalApplication No. 60/453,706, filed Apr. 5, 2002, which was converted fromU.S. Ser. No. 10/116,866, and which is incorporated herein by reference.

This invention was made with government support under CA 74238, CA 82713and CA 30199 awarded by the National Cancer Institute and DAMD17-02-1-0315 and DAMD 17-98-1-8164, both awarded by the Department ofDefense. The government has certain rights in this invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the fields of cancer,molecular medicine and drug delivery and, more specifically, tomolecules that selectively home to tumor blood vessels and tumor cells.

2. Background Information

A major hurdle to advances in treating cancer is the lack of agents thatare effective in selectively targeting a cancer while sparing normaltissue. Radiation therapy and surgery, for example, which generally arelocalized treatments, can cause substantial damage to normal tissue inthe treatment field, resulting in scarring and loss of normal tissue.Chemotherapy, in comparison, which generally is administeredsystemically, can cause substantial damage to organs such as bonemarrow, mucosa, skin and the small intestine, which undergo rapid cellturnover and continuous cell division. As a result, undesirable sideeffects such as nausea, loss of hair and drop in blood cell count occuras a result of systemic treatment with a chemotherapeutic agent. Suchundesirable side effects often limit the amount of drug that can besafely administered, thereby hampering survival rate and impacting thequality of patient life.

Anticancer agents that target DNA are some of the most effective agentsin clinical use and are responsible for significant increases in thesurvival of cancer patients when administered in combination with otherdrugs. Effective anticancer agents that target DNA include alkylatingagents and agents that intercalate into DNA or result in double-strandedDNA breaks. Exemplary DNA-targeted drugs in use or clinical trial todayare cyclophosphamide, melphalan, mitomycin C, bizelesin, cisplatin,doxorubicin, etoposide, mitoxantrone, actinomycin D and bleomycin(Hurley, Nature Reviews Cancer 2:188-200 (2002)). Unfortunately, likemany other anti-cancer agents, DNA-targeted drugs are extremely toxicand result in significant side effects (Slapak and Kufe in Harrison'sPrinciples of Internal Medicine 14^(th) Edition pages 523-537McGraw-Hill, Inc. New York 1998). As an example, use of the platinumagent, cisplatin, can be limited by severe nausea, vomiting, neuropathyand myelosuppression.

Selective delivery of DNA-targeting drugs and other anticancer agents totumor cells or the vasculature that supports tumor growth would resultin less toxic therapy since rapidly proliferating normal cells would bespared. However, to date, it has been difficult to produce drugs thattarget cancer-specific genes or that are delivered specifically tocancer cells or supporting vasculature. Thus, there is a need formolecules that selectively target tumor cells and tumor vasculature. Thepresent invention satisfies this need and also provides relatedadvantages.

SUMMARY OF THE INVENTION

The present invention provides an isolated homing molecule thatselectively homes to tumor blood vessels and tumor cells and thatspecifically binds nucleolin, where the molecule is not a peptide havinga length of more than 85 residues. A homing molecule of the inventioncan be, for example, a peptide or peptidomimetic.

The present invention also provides an isolated peptide orpeptidomimetic having a length of less than 85 residues that containsthe amino acid sequence KDEPQRRSARLSAKPAPPKPEPKPKKAPAKK (SEQ ID NO: 9)or a peptidomimetic of this sequence. In one embodiment, the inventionprovides an isolated peptide having a length of less than 85 residuesthat contains the amino acid sequence KDEPQRRSARLSAKPAPPKPEPKPKKAPAKK(SEQ ID NO: 9). An isolated peptide or peptidomimetic of the inventioncan have, for example, a length of less than 50 residues or a length ofless than 35 residues.

Further provided herein is an isolated homing peptide or peptidomimeticof less than 85 residues that selectively homes to tumor blood vesselsand tumor cells and that specifically binds nucleolin. In oneembodiment, such an isolated homing peptide or peptidomimetic includesthe amino acid sequence KDEPQRRSARLSAKPAPPKPEPKPKKAPAKK (SEQ ID NO: 9)or a conservative variant or peptidomimetic of this sequence. In anotherembodiment, the isolated homing peptide or peptidomimetic of theinvention that specifically binds nucleolin is a peptide. In furtherembodiments, such an isolated homing peptide or peptidomimetic has alength of less than 50 residues or a length of less than 35 residues.

The present invention further provides a conjugate which contains atherapeutic moiety linked to a homing molecule that selectively homes totumor blood vessels and tumor cells and that specifically bindsnucleolin. In one embodiment, such a conjugate contains a homingmolecule which is not an antibody or antigen-binding fragment thereofsuch as an anti-nucleolin antibody or antigen-binding fragment. Inanother embodiment, the conjugate contains a peptide or peptidomimeticportion having a length of at most 200 residues. In a furtherembodiment, the conjugate contains a peptide or peptidomimetic portionhaving a length of at most 50 residues.

A homing molecule incorporated into a conjugate of the invention can be,for example, a homing peptide or peptidomimetic. In one embodiment, aconjugate of the invention includes a homing peptide or peptidomimeticcontaining the amino acid sequence SEQ ID NO: 9 or a conservativevariant or peptidomimetic of this sequence. Such a homing peptide orpeptidomimetic can include, for example, the amino acid sequence SEQ IDNO: 9, or a peptidomimetic thereof. In another embodiment, a conjugateof the invention includes a homing peptide or peptidomimetic whichcontains the amino acid sequence SEQ ID NO: 11 or a conservative variantor peptidomimetic thereof. Such a homing peptide or peptidomimetic caninclude, for example, the amino acid sequence SEQ ID NO: 11, or apeptidomimetic of this sequence.

A variety of therapeutic moieties are useful in the conjugates of theinvention, including, without limitation, anti-angiogenic agents andcytotoxic agents, such as those that target a DNA-associated process. Acytotoxic agent that targets a DNA-associated process can be, forexample, an alkylating agent, an anti-tumor antibiotic or asequence-selective agent. As non-limiting examples, cytotoxic agentsthat target a DNA-associated process encompass cyclophosphamide,melphalan, mitomycin C, bizelesin, cisplatin, doxorubicin, etoposide,mitoxantrone, SN-38, Et-743, actinomycin D, bleomycin and TLK286.

If desired, a conjugate of the invention can be multivalent, includingat least two homing molecules that each selectively homes to tumor bloodvessels and tumor cells and that each specifically blind nucleolin. Inparticular embodiments, a conjugate of the invention includes at leastten or at least 100 of such homing molecules. A variety of therapeuticmoieties are useful in the multivalent conjugates of the inventionincluding, but not limited to, phage moieties.

In a further embodiment, the invention provides a multivalent conjugatecontaining at least two homing peptides or peptidomimetics that eachselectively homes to tumor blood vessels and tumor cells and that eachindependently contains the amino acid sequence SEQ ID NO: 9 or aconservative variant or peptidomimetic of this sequence. In oneembodiment, such a conjugate contains at least ten homing peptides orpeptidomimetics that each selectively homes to tumor blood vessels andtumor cells and that each independently contains the amino acid sequenceSEQ ID NO: 9 or a conservative variant or peptidomimetic thereof. Inanother embodiment, a conjugate of the invention contains at least 100homing peptides or peptidomimetics that each selectively homes to tumorblood vessels and tumor cells and that each independently contains theamino acid sequence SEQ ID NO: 9 or a conservative variant orpeptidomimetic thereof. Any of the above multivalent conjugates of theinvention can include a variety of therapeutic moieties, for example, aphage moiety.

Also provided herein is a conjugate containing a detectable label linkedto a homing molecule that selectively homes to tumor blood vessels andtumor cells and that specifically binds nucleolin. A variety ofdetectable labels are useful in such a conjugate including radionuclidesand fluorescent labels.

The present invention also provides a method of directing a therapeuticmoiety to tumor blood vessels and tumor cells in a subject byadministering to the subject a conjugate which contains a therapeuticmoiety linked to a homing molecule that selectively homes to tumor bloodvessels and tumor cells and that specifically binds nucleolin, therebydirecting the therapeutic moiety to tumor blood vessels and tumor cells.In one embodiment, the homing molecule is not an antibody orantigen-binding fragment thereof. In other embodiments, the peptide orpeptidomimetic portion of the conjugate has a length of at most 200residues, or a length of at most 50 residues.

A variety of homing molecules are useful in the methods of the inventionincluding homing peptides and peptidomimetics. A method of directing atherapeutic moiety to tumor blood vessels and tumor cells in a subjectcan be practiced, for example, using a homing peptide or peptidomimeticthat contains the amino acid sequence SEQ ID NO: 9, or a conservativevariant or peptidomimetic of this sequence. In one embodiment, such ahoming peptide or peptidomimetic includes the amino acid sequence SEQ IDNO: 9, or a peptidomimetic thereof. A method of directing a therapeuticmoiety to tumor blood vessels and tumor cells in a subject also can bepracticed, for example, with a homing peptide or peptidomimetic whichcontains the amino acid sequence SEQ ID NO: 11, or a conservativevariant or peptidomimetic of this sequence. In one embodiment, themethod is practiced with a conjugate containing a homing peptide orpeptidomimetic that includes the amino acid sequence SEQ ID NO: 11 or apeptidomimetic thereof.

A variety of therapeutic moieties can be directed to tumor blood vesselsand tumor cells in a subject according to a method of the invention.Such moieties encompass, without limitation, anti-angiogenic agents andcytotoxic agents, including cytotoxic agents that target aDNA-associated process such as alkylating agents, anti-tumor antibioticsand sequence-selective cytotoxic agents. In particular embodiments, amethod of the invention relies on one of the following cytotoxic agentsthat target a DNA-associated process: cyclophosphamide, melphalan,mitomycin C, bizelesin, cisplatin, doxorubicin, etoposide, mitoxantrone,SN-38, Et-743, actinomycin D, bleomycin or TLK286.

The present invention also provides a method of imaging tumors and tumorvasculature in a subject by administering to the subject a conjugatecontaining a detectable label linked to a homing molecule thatselectively homes to tumor blood vessels and tumor cells and thatspecifically binds nucleolin; and detecting the conjugate, therebyimaging tumors and tumor vasculature. A homing molecule useful in animaging method of the invention can be, for example, a homing peptide orpeptidomimetic such as a homing peptide or peptidomimetic that containsthe amino acid sequence SEQ ID NO: 9 or a conservative variant orpeptidomimetic of this sequence. Any of a variety of detectable labelsare useful in the imaging methods of the invention, includingfluorescent labels and radionuclides such as indium-111, technetium-99,carbon-11, and carbon-13.

The present invention also provides a method of reducing the number oftumor blood vessels in a subject by administering to the subject aconjugate which contains a cytotoxic agent linked to a homing moleculethat selectively homes to tumor blood vessels and tumor cells and thatspecifically binds nucleolin, thereby reducing the number of tumor bloodvessels in the subject. The peptide or peptidomimetic portion of theconjugate can have, for example, a length of at most 200 residues, or alength of at most 50 residues. In one embodiment, a method of theinvention is practiced with a conjugate containing a homing peptide orpeptidomimetic. In a further embodiment, a method of the invention ispracticed with a conjugate containing a homing peptide or peptidomimeticthat includes the amino acid sequence SEQ ID NO: 9, or a conservativevariant or peptidomimetic of this sequence. Any of the therapeuticmoieties described above, such as anti-angiogenic agents, cytotoxicagents and cytotoxic agents that target a DNA-associated process, aswell as additional moieties disclosed herein or known in the art, can beused to reduce the number of tumor blood vessels according to a methodof the invention.

Also provided herein is a method of treating cancer in a subject byadministering to the subject a conjugate which contains a therapeuticmoiety linked to a homing molecule that selectively homes to tumor bloodvessels and tumor cells and that specifically binds nucleolin. Inparticular embodiments, the peptide or peptidomimetic portion of theconjugate has a length of at most 200 residues, or a length of at most50 residues. In other embodiments, a method of the invention ispracticed with a conjugate containing a homing peptide or peptidomimeticsuch as a homing peptide or peptidomimetic that includes the amino acidsequence SEQ ID NO: 9, or a conservative variant or peptidomimetic ofthis sequence. It is understood that, in a method of the invention fortreating cancer in a subject, any of a variety of therapeutic moietiescan be useful, including but not limited to, anti-angiogenic agents;cytotoxic agents; and cytotoxic agents that target a DNA-associatedprocess.

The present invention further provides a method of isolating progenitorcells from a heterogeneous mixture of cells by contacting theheterogenous mixture of cells with a homing molecule that selectivelyhomes to tumor blood vessels and tumor cells and specifically bindsnucleolin under conditions suitable for specific binding of the homingmolecule to the progenitor cells; and separating cells that bind thehoming molecule from non-binding cells, thereby isolating progenitorcells from the heterogenous mixture of cells. The heterogeneous mixtureof cells can be, for example, primary tissue such as primary bonemarrow.

In one embodiment, isolation of progenitor cells according to a methodof the invention is practiced with a homing peptide or peptidomimetic.In a further embodiment, the method is practiced with a homing peptideor peptidomimetic containing the amino acid sequenceKDEPQRRSARLSAKPAPPKPEPKPKKAPAKK (SEQ ID NO: 9) or a conservative variantor peptidomimetic thereof. In another embodiment, the method ispracticed with a homing peptide or peptidomimetic containing the aminoacid sequence SEQ ID NO: 11 or a conservative variant or peptidomimeticthereof. Homing peptides and peptidomimetics useful in isolatingprogenitor cells can have a variety of lengths, including, withoutlimitation, a length of less than 85 residues, a length of less than 50residues, or a length of less than 35 residues.

A method of the invention for isolating progenitor cells can bepracticed, if desired, with a homing molecule attached to a support. Amethod of the invention also can be practiced, for example, with ahoming molecule linked to a fluorescent label. In one embodiment, theseparation step includes fluorescence activated cell sorting (FACS). Infurther embodiments, progenitor cells are isolated using a homingpeptide or peptidomimetic containing the amino acid sequence SEQ ID NO:9 or SEQ ID NO: 11, or a conservative variant or peptidomimetic of oneof these sequences, linked to a fluorescent label.

The present invention also provides a method of isolating one or morehoming molecules that selectively home to tumor blood vessels and tumorcells by contacting nucleolin, or a fragment thereof, with a library ofmolecules under conditions suitable for specific binding of a moleculeto nucleolin; assaying for specific binding; and separating one or morenucleolin-binding molecules from the library, thereby isolating one ormore homing molecules that selectively home to tumor blood vessels andtumor cells. In one embodiment, a screening method of the invention ispracticed by assaying for specific binding to purified nucleolin. Inanother embodiment, a screening method of the invention is practiced byassaying for specific binding to a fragment of nucleolin including theNCL3 domain. In yet another embodiment, a screening method of theinvention is practiced by assaying for specific binding to cellsexpressing nucleolin on the cell surface and further assaying forspecific binding to control cells which do not express cell-surfacenucleolin.

BRIEF DESCRIPTION OF THE DRAWINGS

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FIG. 1 shows phage enrichment in vivo. The Y-axis shows fold enrichmentof selected phage relative to the unselected cDNA phage library pool.

FIG. 2 shows characterization of HMGN2 clones and localization of theHMGN2 cell binding domain. (A) Amino acid sequences of five isolatedhuman HMGN2 clones (SEQ ID NOS: 1 to 5) with SEQ ID NOS indicated inparenthesis to the left. The corresponding portion of human HMGN2 fromGenbank accession M12623 (Swissprot accession P05204) is shown as SEQ IDNO: 6. Intron-exon boundaries of the gene are marked with arrows, andthe sequence encoded by exon 3 of HMGN2 is underlined. (B) The sequenceand cell binding activity of four fragments (SEQ ID NOS: 7 to 10)corresponding to portions of SEQ ID NO: 5. SEQ ID NOS are indicated inparentheses above or below the sequence. Phage binding to primary tumorcells obtained from HL-60 xenograft tumors was assayed; the results arerepresented as fold enrichment relative to non-recombinant T7 phage. Thenumber of plaque forming units (pfu) are indicated above each column.One experiment representative of four is shown.

FIG. 3 shows tissue localization of intravenously injected HMGN2 peptideSEQ ID NO: 9. All panels except panel (E) show immunofluorescence oftissue samples from mice injected with fluorescein-labeled peptide SEQID NO: 9. (A) HL-60 tumor; (B) brain; (C) skin; (D) gut; (E)fluorescein-labeled ARALPSQRSR (SEQ ID NO: 13) control peptide in micebearing HL-60 xenograft; (F) MDA-MB-435 tumor; (G) a highermagnification view from panel A showing the localization of SEQ ID NO: 9(green), lectin stained vasculature (red) and DAPI stained nuclei(blue). Green and blue images are shown individually in panels H and I.Magnification: panels A, B and E, 200×; panels C and D, 100×; and panelsF-I, 400×.

FIG. 4 shows a FACS profile of bone marrow cells labeled withfluorescent peptide SEQ ID NO: 9 and with antibodies against celldifferentiation markers. (A) control peptide SEQ ID NO: 13(number/percentage of cells in lower right quadrant: 1/0.0). (B) peptideSEQ ID NO: 9 (308/0.88). (C) SEQ ID NO: 9 vs. CD45 (77% CD45-positive).(D) SEQ ID NO: 9 vs. CD34 (75% CD34-negative).

FIG. 5 shows uptake and nuclear translocation of peptide SEQ ID NO: 9 incultured tumor cells. HL-60 cells were incubated with (A)fluorescein-labeled SEQ ID NO: 9 or (B) fluorescein-labeled ARALPSQRSR(SEQ ID NO: 13) control peptide, stained with DAPI (blue) and examinedby confocal microscopy. (C-F) MDA-MB-435 cells incubated with peptideSEQ ID NO: 9 synthesized either from (C and D) L amino acids or (E andF) D amino acids, stained with DAPI and examined under an epifluorescentmicroscope. Panels (C) and (E) were analyzed using a green (FITC)filter; panels (D) and (F) were analyzed using a blue (DAPI) filter.Magnification (A) and (B), 400×; C-F, 200×.

FIG. 6 shows that nucleolin binds to immobilized peptide SEQ ID NO: 9.(A) SDS gel electrophoresis of proteins isolated from MDA-MB-435 cellextracts on SEQ ID NO: 9 affinity matrix (“F3”) or control matrix(“control”). The arrow indicates a specific 110-kDa band, which wasidentified as nucleolin by mass spectroscopy. (B) Immunoblotting ofeluates from peptide SEQ ID NO: 9 (“F3”) and control peptide affinitymatrices and unfractionated MDA-MB-435 cell extract (“extract”) with amurine monoclonal anti-nucleolin antibody.

FIG. 7 shows cell surface expression of nucleolin in MDA-MB-435 breastcancer cells. (A) Peptide SEQ ID NO: 9 affinity chromatography (“F3”)and control peptide chromatography of biotin-labeled proteinssolubilized from cell surface-biotinylated MDA-MB-435 cells. (B)Fluorescence activated cell sorting (FACS) analysis of MDA-MB-435 cellsand various antibodies. Propidium iodide-negative (living) cells weregated for the analysis.

FIG. 8 shows that anti-nucleolin antibodies inhibit internalization ofpeptide SEQ ID NO: 9 into MDA-MB-435 cells. Exponentially growing cellswere incubated with 1 μM FITC-SEQ ID NO: 9 (A,B,C) or FITC-LyP1 controlpeptide (D,E) for two hours at 37° C. and co-incubated with NCL3anti-nucleolin antibody (B,D). FITC staining is shown in green; DAPIstaining of nuclei is shown in blue.

FIG. 9 shows that glycosaminoglycan-deficient cells bind and internalizepeptide SEQ ID NO: 9. (A) Binding of SEQ ID NO: 9-displaying phage(“F3”) to glycosaminoglycan-deficient pgsA-745 cells and parental CHO-K1cells. (B) Immunofluorescence of FITC-labeled peptide SEQ ID NO: 9 orcontrol peptide in CHO-K1 or pgsA-745 cells. Panel a: ControlFITC-peptide in CHO-K1 cells. Panel b: FITC-labeled peptide SEQ ID NO: 9in CHO-K1 cells. Panel c: Control FITC-labeled peptide in pgsA-745cells. Panel d: FITC-labeled peptide SEQ ID NO: 9 in pgsA-745 cells.

FIG. 10 shows the subcellular distribution of nucleolin in dividing andstationary cells. MDA-MB-435 cells were stained with NCL3 anti-nucleolinpolyclonal antibody (red) and counter-stained with4′,6-diamidino-2-phenylindole (DAPI; blue) after growth in media with orwithout fetal calf serum and in both intact and permeabilized cells. (A)Fixed, intact cells cultured in standard media (B) Fixed, intact cellswhich were serum starved. (C) Triton X-100-permeabilized cells culturedin standard media. (D) Triton X-100-permeabilized cells which were serumstarved.

FIG. 11 shows that cell surface nucleolin is specific for tumor bloodvessels in vivo. Mice bearing MDA-MB-435 xenograft tumors wereintravenously injected with polyclonal anti-nucleolin antibodies. Tumorand control organs were removed one hour following injection, andsectioned and examined for nucleolin staining. Blood vessels werestained with anti-CD31 antibody (green), and nuclei were counterstainedwith DAPI (blue). (A,B) Tumor blood vessels from mice injected withanti-nucleolin. (C) Blood vessels of the skin from mice injected withanti-nucleolin. (D) Tumor blood vessels from mouse injected with controlrabbit IgG. Magnification: A, C and D, ×200; B, ×400.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to the discovery of homing moleculeswhich selectively home to tumor blood vessels and tumor cells, forexample, to leukemia and breast cancer cells and their vasculature, inpreference to most non-tumor tissue. A homing molecule of the inventionalso can selectively home, as described further below, to cells that actas progenitors of tumor vasculature.

As disclosed herein, a screening strategy was developed to identifymolecules that recognize an epitope shared by endothelial progenitorcells within the bone marrow and by tumor endothelial cells. Bypre-selection for binding to lineage-depleted bone marrow cells(putative progenitor cells) ex vivo and further selection for homing toHL-60 xenograft tumors in vivo, a selected phage pool was producedshowing 20-fold enrichment for tumor homing in vivo relative to theunselected cDNA library (see Example I and FIG. 1). The predominant cDNAin the selected phage pool (SEQ ID NO: 5) was a fragment encoding thefirst 73 residues of the high mobility group protein HMGN2, a highlyconserved nucleosomal protein involved in unfolding higher-orderchromatin structure and facilitating transcriptional activation ofmammalian genes. In addition to the predominant cDNA, additional HMGN2clones isolated from the selected phage pool (SEQ ID NOS: 1 to 4) allshared a common sequence corresponding to exons 3 and 4 of HMGN2 (seeFIG. 2A).

As further disclosed herein, phage displaying a set of sequencescorresponding to fragments of the amino-terminal portion of HMGN2 wereconstructed to localize the HMGN2 domain responsible for cell bindingand in vivo homing. This set of phage was tested for activity in bindingto primary cells from HL-60 xenograft tumors. As shown in FIG. 2B, whenphage bearing fragment SEQ ID NO: 7, 8, 9 or 10 were tested for tumorbinding activity as compared to non-recombinant phage, only phagebearing the 31-amino acid fragment encoded by exons 3 and 4 (SEQ ID NO:9), which corresponds to the nucleosomal binding domain of HMGN2,demonstrated substantial tumor cell binding activity (see Example II).Furthermore, binding of SEQ ID NO: 9-displaying phage to tumor cells wasinhibited by free SEQ ID NO: 9 peptide in a dose dependent fashion,indicating that the phage binding was specific. Furthermore, phageexpressing a sub-sequence of SEQ ID NO: 9 (PQRRSARLSA; SEQ ID NO: 11)exhibited a 90-fold binding preference for tumor cells as compared tonon-recombinant phage.

As further disclosed herein in Example III, fluorescein-conjugated SEQID NO: 9 was injected into the tail vein of mice bearing HL-60 orMDA-MB-435 xenografts. Histological analysis showed a strong fluorescentsignal in tumor tissue, whereas little or no specific fluorescence wasdetected in normal brain, liver or spleen. Within the HL-60 leukemiatumor tissue, SEQ ID NO: 9 localized to tumor cells and cells liningtumor blood vessels, indicating that SEQ ID NO: 9 binding is a sharedproperty of tumor cells and tumor endothelial cells. Furthermore,peptide SEQ ID NO: 9 selectively homed to a variety of tumor types;selective homing was observed with all tumor types tested, includingHL-60 leukemia, MDA-MB-435 breast cancer and TRAMP mouse prostatecarcinoma. In sum, the results disclosed herein with fluorescein andrhodamine conjugates of SEQ ID NO: 9, as well as phage-displayed SEQ IDNO: 9, indicate that this HMGN2-derived peptide can be used to target amoiety such as a drug to tumors and tumor vasculature.

As further disclosed herein, fluorescein-labeled SEQ ID NO: 9accumulated in the nucleus of target cells in vivo and in vitro (seeFIGS. 3 and 5). For example, cultured HL-60 or MDA-MB-435 cellsincubated with 1 μM fluorescein-labeled SEQ ID NO: 9 revealed nuclearpeptide localization within 30 minutes. These results demonstrate thatpeptide SEQ ID NO: 9 localizes to the nuclei of tumor and endothelialcells upon internalization.

Additional results disclosed herein demonstrate that the cell surfacemolecule recognized by HMGN2-derived peptides such as SEQ ID NO: 9 iscell-surface nucleolin and that internalization of peptide SEQ ID NO: 9is entirely dependent on cell surface expression of nucleolin. Asdisclosed in Example VI, affinity chromatography of MDA-MB-435 cellextracts revealed a major band of about 110 kDa, which was identified byspectrometric analysis as nucleolin (FIG. 6A).

Furthermore, anti-nucleolin antibodies and cell-surface biotin labelingindicated that nucleolin is expressed on the surface of actively growingcells, but is exclusively nuclear in serum-starved non-dividing cells(see FIG. 10). Thus, cell surface expression of nucleolin is associatedwith active cell proliferation. Both uptake of peptide SEQ ID NO: 9 andstaining of intact cells with anti-nucleolin antibodies were suppressedin serum-starved cells. In addition, HL-60 leukemia cells induced todifferentiate in culture in non-proliferating macrophage lose theability to internalize peptide SEQ ID NO: 9. As further disclosedherein, nucleolin was expressed at the cell surface in tumor vasculaturein vivo (FIG. 11). These results indicate that exprssion of cell surfacenucleolin and the ability to bind and internalize HMGN2-derived peptidessuch as SEQ ID NO: 9 is much more restricted in vivo than in vitro.These results demonstrate that HMGN2-derived peptides such as peptideSEQ ID NO: 9 and other molecules that bind nucleolin can be useful forselectively targeting anti-angiogenic agents or other anti-cancertherapeutics into the nucleus of tumor cells as well as tumorendothelial cells.

Thus, the present invention relates, in part, to the surprisingdiscovery that a peptide derived from HMGN2, which is one of the highmobility group (HMG) proteins, can selectively accumulate in tumors andtumor vasculature upon intravenous administration. HMGN2 (HMG-17) is arelatively abundant protein expressed in the nuclei of all highereukaryotes, that functions in unfolding of higher-order chromatinstructure and in facilitating transcriptional activation in mammals(Bustin, Mol. Cell. Biol. 19:5237-5246 (1999)). As a group, the HMGproteins are abundant, ubiquitous proteins that bind to DNA in asequence-independent manner. The HMG proteins can be divided into threesubfamilies, the HMG-1/2 subfamily; the HMG-I/Y subfamily and theHMG-14/17 subfamily, each of which have a characteristic functionalsequence motif which is the main site of interaction between the HMGprotein and the DNA or chromatin target (Bustin, supra, 1999).

HMGN2 belongs to the HMG-14/17 subfamily, which contains HMG proteinscharacterized by a nucleosomal binding domain that specificallyrecognizes the generic structure of the 146 bp nucleosome core (Bustinand Reeves, Prog. Nucl. Acids Res. Mol. Biol. 54:35-100 (1996)). HMGN2binds to nucleosomes cooperatively via the nucleosomal binding domain toform a homodimeric complex, and the carboxy terminal region of HMGN2mediates changes in chromatin structure (Ding et al., Mol. Cell. Biol.17:5843-5855 (1997); Trieschmann et al., Mol. Cell. Biol. 15:6663-6669(1995)). The major sites of interaction between HMGN2 and thenucleosomal core DNA are located 25 bp from the end of the DNA and inthe two major grooves flanking the nucleosomal dyad axis (Alfonso etal., J. Mol. Biol. 236:189-198 (1994)). The nucleosomal binding domainmotif is a positively charged stretch of approximately 30 amino acidswith a bipartite structure: the highly conserved amino-terminal regionof the nucleosomal binding domain is enriched in arginine residues,while the carboxy-terminal region contains a preponderance of lysine andproline (Bustin and Reeves, supra, 1996).

HMGN2 functions to enhance transcription and replication, although onlyfrom chromatin and not from DNA templates, indicating that this proteinacts as a modifier of chromatin structure rather than as apolymerase-specific factor. Enhancement of DNA-dependent activities isassociated with decompaction of the nucleosome array in the chromatinfiber; both transcriptional regulation and chromatin decompaction aremediated by the negatively charged C-terminal domain of HMGN2 (Ding etal., supra, 1996; Trieschmann et al., supra, 1995). This C-terminaldomain contacts the amino-terminal tail of histone H3, near the lysineresidues serving as targets for histone acetyltransferases, and alsotargets histone H1.

Based on the above findings, the present invention provides an isolatedpeptide or peptidomimetic having a length of less than 85 residues thatcontains the amino acid sequence KDEPQRRSARLSAKPAPPKPEPKPKKAPAKK (SEQ IDNO: 9) or a peptidomimetic of this sequence. In one embodiment, theinvention provides an isolated peptide having a length of less than 85residues that contains the amino acid sequence SEQ ID NO: 9. An isolatedpeptide or peptidomimetic of the invention can have, for example, alength of less than 50 residues or a length of less than 35 residues.

The invention also provides an isolated homing peptide or peptidomimeticof less than 85 residues that selectively homes to tumor blood vesselsand tumor cells and contains the amino acid sequence SEQ ID NO: 9 or aconservative variant or peptidomimetic of this sequence. In oneembodiment such an isolated homing peptide or peptidomimetic is apeptide. In other embodiments, the peptide or peptidomimetic has alength of less than 50 residues or a length of less than 35 residues.

The peptides and peptidomimetics of the invention are provided inisolated form. As used herein in reference to a peptide orpeptidomimetic of the invention, the term “isolated” means a peptide orpeptidomimetic that is in a form that is relatively free from materialsuch as contaminating polypeptides, lipids, nucleic acids and othercellular material that normally is associated with the peptide orpeptidomimetic in a cell or that is associated with the peptide orpeptidomimetic in a library or in a crude preparation.

The peptides and peptidomimetics of the invention, including thebifunctional, multivalent and homing peptides and peptidomimeticsdiscussed below, can have a variety of lengths. A peptide orpeptidomimetic of the invention can have, for example, a relativelyshort length of less than eight, nine, ten, 12, 15, 20, 25, 30, 35, 40,45, 50, 60, 70 or 80 residues. A peptide or peptidomimetic of theinvention also can be useful in the context of a significantly longersequence as described further below. As used herein, the term “residue”refers to amino acids or analogs thereof. It is understood that apeptide containing, for example, the amino acid sequence SEQ ID NO: 9includes the specified amino acids as a contiguous sequence notseparated by other amino acids.

The present invention also provides an isolated peptide orpeptidomimetic containing an amino acid sequence which is a conservativevariant, for example, of the sequence KDEPQRRSARLSAKPAPPKPEPKPKKAPAKK(SEQ ID NO: 9). As used herein, a “conservative variant” is an aminoacid sequence in which a first amino acid is replaced by a second aminoacid or amino acid analog having at least one similar biochemicalproperty, which can be, for example, similar size, charge,hydrophobicity or hydrogen-bonding capacity. For example, a firsthydrophobic amino acid can be conservatively substituted with a second(non-identical) hydrophobic amino acid such as alanine, valine, leucine,or isoleucine, or an analog thereof. Similarly, a first basic amino acidcan be conservatively substituted with a second basic amino acid such asarginine or lysine, or an analog thereof. In the same way, a firstacidic amino acid can be conservatively substituted with a second acidicamino acid such as aspartic acid or glutamic acid, or an analog thereof,or an aromatic amino acid such as phenylalanine can be conservativelysubstituted with a second aromatic amino acid or amino acid analog, forexample, tyrosine.

As disclosed herein, a peptide or peptidomimetic of the invention canmaintain homing activity in the context of a significantly longersequence. For example, the 31-mer peptideKDEPQRRSARLSAKPAPPKPEPKPKKAPAKK (SEQ ID NO: 9) maintained the ability tohome when fused to a phage coat protein, confirming that a peptide ofthe invention can have selective homing activity when embedded in alarger protein sequence. Thus, the invention further provides a chimericprotein containing a peptide or peptidomimetic of the invention, or ahoming peptide or peptidomimetic of the invention, fused to aheterologous protein. In one embodiment, the invention provides achimeric protein containing a homing peptide or peptidomimetic thatselectively homes to tumor blood cells or tumor cells and thatspecifically binds nucleolin fused to a heterologous protein. In oneembodiment, the heterologous protein has a therapeutic activity. In afurther embodiment, the heterologous protein is an antibody orantigen-binding fragment thereof. In other embodiments, the inventionprovides a chimeric protein in which a peptide or peptidomimeticcontaining the amino acid sequence SEQ ID NO: 9, or a conservativevariant or peptidomimetic of this sequence, is fused to a heterologousprotein. The term “heterologous,” as used herein in reference to aprotein fused to a peptide or peptidomimetic of the invention, means aprotein derived from a source other than the gene encoding the peptideof the invention or upon which the peptidomimetic is derived. A chimericprotein of the invention can have a variety of lengths, for example, upto 100, 200, 300, 400, 500, 800, 1000 or 2000 residues or more.

The invention also provides a bifunctional peptide which contains ahoming peptide that selectively homes to tumor blood cells or tumorcells and that specifically binds nucleolin, fused to a second peptidehaving a separate function. Such bifunctional peptides have at least twofunctions conferred by different portions of the peptide and can, forexample, display anti-angiogenic activity or pro-apoptotic activity inaddition to selective homing activity. As a non-limiting example, theinvention provides a bifunctional peptide having the sequenceKDEPQRRSARLSAKPAPPKPEPKPKKAPAKK-GG-_(D)(KLAKLAK)₂ (SEQ ID NO: 21).Insuch a peptide, the KDEPQRRSARLSAKPAPPKPEPKPKKAPAKK (SEQ ID NO: 9)portion exhibits selective homing activity, while the _(D)(KLAKLAK)₂(SEQ ID NO: 14)portion exhibits pro-apoptotic activity.

The present invention further provides an isolated multivalent peptideor peptidomimetic that includes at least two motifs each independentlycontaining the amino acid sequence SEQ ID NO: 9, or a conservativevariant or peptidomimetic thereof. The multivalent peptide orpeptidomimetic can have, for example, at least three, at least five orat least ten of such motifs, each independently containing the aminoacid sequence SEQ ID NO: 9, or a conservative variant or peptidomimeticthereof. In particular embodiments, the multivalent peptide orpeptidomimetic has two, three, four, five, six, seven, eight, nine, ten,fifteen or twenty identical or non-identical motifs of the amino acidsequence SEQ ID NO: 9, or a conservative variant or peptidomimeticthereof. In another embodiment, the multivalent peptide orpeptidomimetic contains identical motifs, which consist of the aminoacid sequence SEQ ID NO: 9, or a conservative variant or peptidomimeticof this sequence. In a further embodiment, the multivalent peptide orpeptidomimetic contains contiguous motifs, which can be identical ornon-identical.

Thus, the invention provides peptides and peptidomimetics, includingbifunctional and multivalent peptides and peptidomimetics, and homingpeptides and peptidomimetics as discussed further below. As used herein,the term “peptide” is used broadly to mean peptides, proteins, fragmentsof proteins and the like. The term “peptidomimetic,” as used herein,means a peptide-like molecule that has the activity of the peptide uponwhich it is structurally based. Such peptidomimetics include chemicallymodified peptides, peptide-like molecules containing non-naturallyoccurring amino acids, and peptoids, and have an activity such asselective homing activity of the peptide upon which the peptidomimeticis derived (see, for example, Goodman and Ro, Peptidomimetics for DruqDesign, in “Burger's Medicinal Chemistry and Drug Discovery” Vol. 1 (ed.M. E. Wolff; John Wiley & Sons 1995), pages 803-861).

A variety of peptidomimetics are known in the art including, forexample, peptide-like molecules which contain a constrained amino acid,a non-peptide component that mimics peptide secondary structure, or anamide bond isostere. A peptidomimetic that contains a constrained,non-naturally occurring amino acid can include, for example, anα-methylated amino acid; α,α-dialkylglycine or α-aminocycloalkanecarboxylic acid; an N^(α)-C^(α) cyclized amino acid; an N^(α)-methylatedamino acid; a β- or γ-amino cycloalkane carboxylic acid; anα,β-unsaturated amino acid; a β,β-dimethyl or β-methyl amino acid; aβ-substituted-2,3-methano amino acid; an N-C^(δ) or C^(α)-C^(δ) cyclizedamino acid; a substituted proline or another amino acid mimetic. Apeptidomimetic which mimics peptide secondary structure can contain, forexample, a nonpeptidic β-turn mimic; γ-turn mimic; mimic of β-sheetstructure; or mimic of helical structure, each of which is well known inthe art. A peptidomimetic also can be a peptide-like molecule whichcontains, for example, an amide bond isostere such as a retro-inversomodification; reduced amide bond; methylenethioether ormethylene-sulfoxide bond; methylene ether bond; ethylene bond; thioamidebond; trans-olefin or fluoroolefin bond; 1,5-disubstituted tetrazolering; ketomethylene or fluoroketomethylene bond or another amideisostere. One skilled in the art understands that these and otherpeptidomimetics are encompassed within the meaning of the term“peptidomimetic” as used herein.

Methods for identifying a peptidomimetic are well known in the art andinclude, for example, the screening of databases that contain librariesof potential peptidomimetics. For example, the Cambridge StructuralDatabase contains a collection of greater than 300,000 compounds thathave known crystal structures (Allen et al., Acta Crystallogr. SectionB, 35:2331 (1979)). This structural depository is continually updated asnew crystal structures are determined and can be screened for compoundshaving suitable shapes, for example, the same shape as a peptide of theinvention, as well as potential geometrical and chemical complementarityto a target molecule. Where no crystal structure of a peptide of theinvention is available, a structure can be generated using, for example,the program CONCORD (Rusinko et al., J. Chem. Inf. Comput. Sci. 29:251(1989)). Another database, the Available Chemicals Directory (MolecularDesign Limited, Informations Systems; San Leandro Calif.), containsabout 100,000 compounds that are commercially available and also can besearched to identify potential peptidomimetics of a peptide of theinvention, for example, with activity in selectively homing to tumorblood vessels and tumor cells.

An isolated peptide or peptidomimetic of the invention, or a homingpeptide, peptidomimetic or molecule of the invention as discussedfurther below, can be cyclic, or otherwise conformationally constrained.As used herein, a “conformationally constrained” molecule, such as apeptide or peptidomimetic, is one in which the three-dimensionalstructure is maintained substantially in one spatial arrangement overtime. Conformationally constrained molecules can have improvedproperties such as increased affinity, metabolic stability, membranepermeability or solubility. Methods of conformational constraint arewell known in the art and include cyclization.

As used herein in reference to a peptide or peptidomimetic, the termcyclic refers to a structure including an intramolecular bond betweentwo non-adjacent amino acids or amino acid analogues. The cyclizationcan be effected through a covalent or non-covalent bond. Intramolecularbonds include, but are not limited to, backbone to backbone, side-chainto backbone and side-chain to side-chain bonds. Methods of cyclizationinclude formation of a disulfide bond between the side-chains ofnon-adjacent amino acids or amino acid analogs; formation of a lactambond, for example, between a side-chain group of one amino acid oranalog thereof to the N-terminal amine of the amino-terminal residue;and formation of lysinonorleucine and dityrosine bonds.

Active fragments of the homing peptide disclosed herein as SEQ ID NO: 9also can be useful in the conjugates and methods of the invention. Asused herein in reference to a peptide sequence such as SEQ ID NO: 9, theterm “active fragment” means a fragment that has substantially the aminoacid sequence of a portion of the 31-amino acid peptide SEQ ID NO: 9 andthat retains substantially the selective homing activity of the parentpeptide. Selective homing activity can be assayed by routine methods, asdescribed in the Examples below. In one embodiment, an active fragmentcontains the amino acid sequence of a portion of SEQ ID NO: 9. Such anactive fragment can have, for example, the amino acid sequence of atleast 10, 12, 15, 18, 20, 22, 25, or 28 contiguous residues of SEQ IDNO: 9.

As disclosed herein, peptide SEQ ID NO: 9 recognizes a target “receptor”which is expressed on tumor cells as well as blood vessel cells oftumors but which is not significantly expressed or available for bindingin most normal tissues. The cell surface and cell-type selectiveexpression of the target receptor, which is disclosed herein asnucleolin, form the basis for the selective homing activity of peptideSEQ ID NO: 9 and related peptides, peptidomimetics and other molecules.Based on this discovery, it is clear that molecules structurallyunrelated to SEQ ID NO: 9 but which also bind cell surface nucleolinalso have the same characteristic of selectively homing to tumor bloodvessels and tumor cells. Such molecules can be identified by the abilityto specifically bind to, or compete for binding to, purified nucleolin,or to compete with SEQ ID NO: 9 for binding to nucleolin-expressingcells such as MDA-MB-435 cells, as described further below. Selectivehoming to tumor blood vessels and tumor cells readily can be confirmedusing in vivo panning as disclosed herein in Example I (see, also, U.S.Pat. No. 5,622,699).

Thus, the present invention provides a method of reducing the number oftumor blood vessels in a subject by administering to the subject ananti-nucleolin antibody or antigen-binding fragment thereof, which isinternalized by tumor endothelial cells. Anti-nucleolin antibodiesuseful in the invention include, without limitation, monoclonalantibodies, humanized antibodies and antibodies against an acidicportion of the amino-terminal domain of nucleolin, which general classesare not mutually exclusive.

The invention further provides a method of treating cancer in a subjectby administering to the subject an anti-nucleolin antibody orantigen-binding fragment thereof, which is internalized by tumor cells.Anti-nucleolin antibodies useful for treating cancer according to amethod of the invention include, without limitation, monoclonalantibodies, humanized antibodies and antibodies against an acidicportion of the amino-terminal domain of nucleolin.

Nucleolin, an abundant nucleolar protein that plays an important role inribosome biogenesis, was initially known as C23 (Orrick et al., Proc.Natl. Acad. Sci., USA 70:1316-1320 (1973)). This ubiquitous protein isencoded by a gene on chromosome 12 with the characteristic GC-richpromoter sequences found in housekeeping genes. Nucleolin regulatesribosome biogenesis and maturation, has a demonstrated helicaseactivity, and also has been implicated in chromatin decondensation,cytoplasmic/nuclear transport of ribosomal components and pre-ribosomalparticles, cytokinesis, replication, embryogenesis and nucleogenesis.Nucleolin also appears to bind specifically to several nuclear proteins,such as nucleophosmin (B23), topoisomerase 1 and the growth factormidkine (Ginisty et al., J. Cell Science 112:761-772 (1999)).

Nucleolin has an apparent molecular weight of 100 to 110 kDa, andnucleolin cDNAs are predicted to encode proteins of about 713 aminoacids. In addition to the human sequence, homologs have been identifiedin hamster, rat, mouse and chicken (LaPeyre et al., Proc. Natl. Acad.Sci. Usa 84:1472-1476 (1987); Srivastava et al., FEBS Letters 250:99-105(1989); Bourbon et al., J. Mol. Biol. 200:627-638 (1988); and Maridorand Nigg, Nucleic Acids Res. 18:1286 (1990)). In nature, nucleolin ishighly phosphorylated and methylated and may be ADP-ribosylated.Analysis of the amino acid sequence of nucleolin reveals threestructural domains. The amino-terminal domain, which controls rRNAtranscription, is made up of highly acidic regions interspersed withbasic sequences and contains multiple phosphorylation sites. Acidicα-helical structures within this domain may bind histone H1. The centralglobular domain, which contains four RNA-binding domains known as RBD orRRM, controls pre-RNA processing. The carboxy-terminal domain, denotedthe GAR or RGG domain, is rich in glycine, arginine and phenylalanineresidues, contains high levels of N^(G),N^(G)-dimethylarginines andfunctions in nucleolar localization (Ginisty et al., supra, 1999).

As used herein, the term “nucleolin” means a polypeptide havingsubstantially the amino acid sequence of a known nucleolin such ashuman, murine, rat, hamster or chicken nucleolin. As a non-limitingexample, nucleolin can have substantially the amino acid sequence ofhuman nucleolin (SEQ ID NO: 19). As described above, a full-lengthnucleolin includes an amino-terminal acidic domain and a centralglobular domain. One skilled in the art appreciates that a fragment of anucleolin polypeptide, for example, retaining one or more amino-terminalacidic stretches, also can be useful in generating internalizinganti-nucleolin antibodies or in screening for homing molecules thatselectively home to tumor cells or tumor vasculature, as describedhereinbelow.

The term nucleolin encompasses a polypeptide having the sequence of anaturally occurring human nucleolin (SEQ ID NO: 19), and includesrelated polypeptides having substantial amino acid sequence similarityto SEQ ID NO: 19. Such related polypeptides typically exhibit greatersequence similarity to nucleolin than to other helicases or nucleolarproteins and include, but are not limited to, species homologies andisotype variants. The term nucleolin generally describes polypeptideshaving an amino acid sequence having greater than about 40% amino acidsequence identity with human nucleolin (SEQ ID NO: 19). In particular, anucleolin can have greater than 50% amino acid identity, greater than60% amino acid identity, greater than 70% amino acid identity, greaterthan 80% amino acid identity, or greater than 85%, 90% or 95% amino acididentity with the human nucleolin sequence SEQ ID NO: 19.

Anti-nucleolin antibodies can be useful as homing molecules in theconjugates and methods of the invention and further can be useful inunconjugated form as anti-tumor and anti-angiogenic agents. As usedherein, the term “antibody” is used in its broadest sense to includepolyclonal and monoclonal antibodies, as well as polypeptide fragmentsof antibodies that retain binding activity for nucleolin of at leastabout 1×10⁵ M⁻¹. One skilled in the art understands that anti-nucleolinantibody fragments including, without limitation, Fab, F(ab′)₂ and Fvfragments, can retain binding activity for nucleolin and, thus, areincluded within the definition of antibody. In addition, the term“antibody,” as used herein, encompasses non-naturally occurringantibodies and fragments usually containing, at a minimum, one V_(H) andone V_(L) domain, such as chimeric antibodies, humanized antibodies andsingle chain Fv fragments (scFv) that specifically or selectively bindnucleolin. Such non-naturally occurring antibodies can be constructedusing solid phase peptide synthesis, produced recombinantly or obtained,for example, by screening phage-displayed or other combinatoriallibraries such as those consisting of variable heavy and light chains asdescribed in Borrebaeck (Ed.), Antibody Engineering (Second edition) NewYork: Oxford University Press (1995)) using, for example, an assaydescribed herein below.

Anti-nucleolin antibodies also can be prepared using a nucleolin fusionprotein or a synthetic peptide encoding a portion of nucleolin such asthe NCL3 domain or another acidic portion of the amino-terminal regionof nucleolin as an immunogen (see Example VI). One skilled in the artunderstands that purified human or other nucleolin, which can beproduced recombinantly, for example, using the human nucleolin nucleicacid sequence disclosed herein as SEQ ID NO: 18, or full-length orfragments of nucleolin, including peptide portions of nucleolin such assynthetic peptide fragments of the human nucleolin amino acid sequencedisclosed herein as SEQ ID NO: 19, can be used as immunogens. It isunderstood that fragments of nucleolin useful as immunogens include theNCL3 domain and related and different fragments of nucleolin that serveto produce nucleolin antibodies which are readily internalized intocells expressing cell-surface nucleolin. One skilled in the art furtherunderstands that non-immunogenic fragments or synthetic peptides ofnucleolin can be made immunogenic by coupling the hapten to a carriermolecule such as bovine serum albumin (BSA) or keyhole limpet hemocyanin(KLH). In addition, various other carrier molecules and methods forcoupling a hapten to a carrier molecule are well known in the art asdescribed, for example, by Harlow and Lane, Antibodies: A LaboratoryManual (Cold Spring Harbor Laboratory Press, 1988)).

Anti-nucleolin antibodies useful in the invention further encompasscommercially available and other anti-nucleolin antibodies known in theart such as, without limitation, the murine anti-human nucleolinmonoclonal 4E2 from AMS Biotechnology Ltd. (United Kingdom) or ResearchDiagnostics Inc. (Flanders, N.J.); the MS3 anti-human monoclonal (U.S.Pat. No. 4,902,615); and the D3 anti-human monoclonal (Deng et al., Mol.Biol. Rep. 23:191-195 (1996); and Hovanessian et al., Exp. Cell Res.261:312-328 (2000)).

Thus, it is clear that a homing molecule useful in the invention can bean HMGN2-derived peptide or an unrelated nucleolin-binding molecule suchas an anti-nucleolin antibody, midkine or basic fibroblast growth factor(bFGF), or a fragment, peptide or peptidomimetic derived therefrom. Asan example, an antibody that binds to the acidic domain at theamino-terminus of nucleolin (NCL3) is internalized by cells, whereas anantibody that binds to another site on nucleolin is not, indicating thatan anti-NCL3 or related similar anti-nucleolin antibody can be usefulfor targeting and internalizing linked therapeutic agents.

Midkine is a 13 kDa cytokine related to pleiotropin which plays a rolein neurite outgrowth and neuronal differentiation and is overexpressedin some human carcinomas. Midkine appears to bind nucleolin through theRGG domain or negatively charged amino-terminal domain. Like thenucleolin-binding peptide SEQ ID NO: 9, midkine is highly basic andbinds to heparin sulfate. One skilled in the art understands thatfull-length midkine or bFGF or fragments or peptidomimetics derived fromthat retain the ability to bind nucleolin also can selectively home totumor vasculature and tumor cells in the same manner as disclosed hereinfor the HMGN2-derived peptide, SEQ ID NO: 9. In one embodiment, theinvention is practiced with a midkine or bFGF-derived peptide orpeptidomimetic with nucleolin-binding activity but without the cytokineactivity of native midkine or bFGF.

Also provided herein is a method of isolating one or more homingmolecules that selectively home to tumor blood vessels and tumor cellsby contacting nucleolin, or a fragment thereof, with a library ofmolecules under conditions suitable for specific binding of a moleculeto nucleolin; assaying for specific binding; and separating one or morenucleolin-binding molecules from the library, thereby isolating one ormore homing molecules that selectively home to tumor blood vessels andtumor cells. Cells that express nucleolin on the cell surface as well aspurified nucleolin, or a fragment thereof, can be useful in thescreening methods of the invention. As non-limiting examples, native,recombinant and human nucleolin, and fragments of human nucleolin suchas the amino-terminal acidic domain (NCL3) and other SEQ ID NO:9-binding fragments of nucleolin, whether purified or expressed on thesurface of a cell, can be useful in the screening methods of theinvention. Libraries that can be screened according to a method of theinvention include, but are not limited to, libraries of peptides andpeptidomimetics, libraries of small molecules, and libraries ofantibodies and antigen-binding fragments thereof, including synthetic,single-chain or other antibody libraries. In one embodiment, a method ofthe invention includes a further step of assaying for internalization ofone or more molecules of the library into a cell expressing cell surfacenucleolin or a fragment thereof. As an example, where the library is alibrary of antibodies, the method can further include assaying forinternalization of one or more nucleolin-binding antibodies into a cellexpressing cell-surface nucleolin or a fragment thereof. Where afragment of nucleolin is utiliized in place of full-lenght nucleolin, itis understood that such a nucleolin fragment is a fragment sufficientfor internalization of a nucleolin-binding molecule such as peptide SEQID NO: 9 or an anti-NCL3 antibody.

Based on the restricted cell surface expression of nucleolin and tumorvessels and tumor cells in vivo, the present invention provides methodsof selectively directing an anti-cancer or anti-angiogenic agent totumor vessels and tumor cells by administering a nucleolin-bindingmolecule linked to the anti-cancer or anti-angiogenic agent. Theinvention also provides a method of treating cancer by administering acytotoxic agent linked to a nucleolin-binding molecule, therebydestroying tumor endothelial cell precursors. Further provided hereinare methods of treating cancer and methods of reducing tumorangiogenesis by reducing cell surface expression of nucleolin.

The present invention also provides an isolated homing molecule thatselectively homes to tumor blood vessels and tumor cells and thatspecifically binds nucleolin, where the molecule is not a peptide havinga length of more than 85 residues. A homing molecule of the inventioncan be, for example, a peptide or peptidomimetic.

The invention also provides an isolated homing peptide or peptidomimeticof less than 85 residues that selectively homes to tumor blood vesselsand tumor cells and that specifically binds nucleolin. In oneembodiment, such an isolated homing peptide or peptidomimetic includesthe amino acid sequence KDEPQRRSARLSAKPAPPKPEPKPKKAPAKK (SEQ ID NO: 9)or a conservative variant or peptidomimetic of this sequence. In anotherembodiment, the isolated homing peptide or peptidomimetic of theinvention that specifically binds nucleolin is a peptide. In furtherembodiments, such an isolated homing peptide or peptidomimetic has alength of less than 50 residues or a length of less than 35 residues.

As used herein, the term “molecule” is used broadly to mean a polymericor non-polymeric organic chemical such as a small molecule drug; anucleic acid molecule such as an RNA, a cDNA or an oligonucleotide; apeptide or peptidomimetic; or a protein such as an antibody or a growthfactor receptor or a fragment thereof such as an Fv, Fd, or Fab fragmentof an antibody containing the antigen-binding domain.

The term “homing molecule” as used herein, means any molecule thatselectively localizes in vivo to the tumor blood vessels and tumor cellsof one or more tumors in preference to most non-tumor tissues.Similarly, the term “homing peptide” or “homing peptidomimetic” means apeptide or peptidomimetic that selectively localizes in vivo to thetumor blood vessels and tumor cells of one or more tumors in preferenceto most non-tumor tissues. It is understood that a homing molecule thatselectively homes in vivo to tumor blood vessels and tumor cells canhome to all tumor types and their supporting blood vasculature or canexhibit preferential homing to the blood vessels and tumor cells of asubset of tumor types.

By “selectively homes” is meant that, in vivo, the homing molecule,peptide or peptidomimetic binds preferentially to tumor blood vesselsand tumor cells, such as leukemias and breast carcinomas and theirsupporting blood vasculature, as compared to most non-tumor tissue.Selective homing generally is characterized by at least a two-foldgreater localization within tumor blood vessels and tumor cells, such assuch as leukemias and breast carcinomas and their supporting bloodvasculature, as compared to a non-tumor tissue such as brain, spleen andliver tissue. A homing molecule can be characterized by 5-fold, 10-fold,20-fold or more preferential localization to tumor blood vessels andtumor cells as compared to brain, spleen or liver, or as compared tomany or most non-tumor tissues. As disclosed herein, the homing moleculeSEQ ID NO: 9 selectively homed to a small population of cells withinskin, gut and bone marrow, and it is understood that a homing moleculecan home, in part, to one or more non-tumor tissues or to a smallpopulation of cells within one or more non-tumor tissues in addition toselectively homing to tumor blood vessels and tumor cells.

A homing molecule of the invention specifically binds nucleolin. As usedherein, the term “specifically binds” or “specifically binding” meansbinding that is measurably different from a non-specific interaction.Specific binding can be measured, for example, by determining binding ofa molecule compared to binding of a control molecule, which generally isa molecule of similar structure that does not have binding activity. Inthis case, specific binding is indicated if the molecule has measurablyhigher affinity for cells expressing cell-surface nucleolin, forexample, HL-60 or MDA-MB-435 cells, than for cells that do not expresscell-surface nucleolin. Specificity of binding can be determined, forexample, by competitive inhibition of the binding of a known bindingmolecule such as SEQ ID NO: 9 or SEQ ID NO: 11.

The term “specifically binding,” as used herein, includes both low andhigh affinity specific binding. Specific binding can be exhibited, forexample, by a low affinity homing molecule having a Kd of at least about10⁻⁴ M. For example, if nucleolin has more than one binding site, ahoming molecule having low affinity can be useful for targeting tumorblood vessels and tumor cells. Specific binding also can be exhibited bya high affinity homing molecule, for example, a homing molecule having aKd of at least about 10⁻⁵ M. Such a molecule can have, for example, a Kdof at least about 10⁻⁶ M, at least about 10⁻⁷ M, at least about 10⁻⁸ M,at least about 10⁻⁹ M, at least about 10⁻¹⁰ M, or can have a Kd of atleast about 10⁻¹¹ M or 10⁻¹² M or greater. Both low and high affinityhoming molecules are useful and are encompassed by the invention. Lowaffinity homing molecules are useful in targeting, for example,multivalent conjugates such as viruses and other particles. Highaffinity homing molecules are useful in targeting, for example,multivalent and univalent conjugates.

The invention further provides a conjugate which contains a therapeuticmoiety linked to a homing molecule that selectively homes to tumor bloodvessels and tumor cells and that specifically binds nucleolin. In oneembodiment, such a conjugate contains a homing molecule which is not anantibody or antigen-binding fragment thereof. In another embodiment, thepeptide or peptidomimetic portion of the conjugate has a length of atmost 200 residues. In a further embodiment, the peptide orpeptidomimetic portion of the conjugate has a length of at most 50residues.

A homing molecule incorporated into a conjugate of the invention can be,for example, a homing peptide or peptidomimetic. In one embodiment, aconjugate of the invention includes a homing peptide or peptidomimeticcontaining the amino acid sequence SEQ ID NO: 9 or a conservativevariant or peptidomimetic of this sequence. Such a homing peptide orpeptidomimetic can include, for example, the amino acid sequence SEQ IDNO: 9, or a peptidomimetic thereof. In another embodiment, a conjugateof the invention includes a homing peptide or peptidomimetic whichcontains the amino acid sequence SEQ ID NO: 11 or a conservative variantor peptidomimetic thereof. Such a homing peptide or peptidomimetic caninclude, for example, the amino acid sequence SEQ ID NO: 11, or apeptidomimetic of this sequence.

A variety of therapeutic moieties are useful in the conjugates of theinvention, including, without limitation, anti-angiogenic agents andcytotoxic agents, such as those that target a DNA-associated process. Acytotoxic agent that targets a DNA-associated process can be, forexample, an alkylating agent, an anti-tumor antibiotic or asequence-selective agent. As non-limiting examples, cytotoxic agentsthat target a DNA-associated process encompass cyclophosphamide,melphalan, mitomycin C, bizelesin, cisplatin, doxorubicin, etoposide,mitoxantrone, SN-38, Et-743, actinomycin D, bleomycin and TLK286.

Also provided herein is a conjugate containing a detectable label linkedto a homing molecule that selectively homes to tumor blood vessels andtumor cells and that specifically binds nucleolin. A variety ofdetectable labels are useful in such a conjugate including radionuclidesand fluorescent labels.

In one embodiment, a conjugate of the invention includes a homingmolecule that is not an antibody or antigen-binding fragment thereof.“Antibody” is an art-recognized term that refers to a peptide orpolypeptide containing one or more complementarity determining regions(CDRs). See, for example, Borrabaeck, Antibody Engineering 2nd Edition,Oxford University Press, New York (1995).

In another embodiment, the peptide or peptidomimetic portion of theconjugate has a defined length. The peptide or peptidomimetic portion ofthe conjugate can have, for example, a length of at most 10, 20, 30, 40,50, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000 or 2000residues. It is understood that the term “peptide or peptidomimeticportion of the conjugate” means total number of residues in the homingpeptide or peptidomimetic and any contiguous protein, peptide orpeptidomimetic, such as a therapeutic protein or pro-apoptotic peptide.

If desired, a conjugate of the invention can be multivalent, includingat least two homing molecules that each selectively homes to tumor bloodvessels and tumor cells and that specifically binds nucleolin. Inparticular embodiments, a multivalent conjugate of the inventionincludes at least ten or at least 100 of such homing molecules. Avariety of therapeutic moieties are useful in the multivalent conjugatesof the invention including, but not limited to, phage moieties.

In a further embodiment, the invention provides a multivalent conjugatecontaining at least two homing peptides or peptidomimetics that eachselectively homes to tumor blood vessels and tumor cells and that eachindependently contains the amino acid sequence SEQ ID NO: 9 or aconservative variant or peptidomimetic of this sequence. In oneembodiment, such a conjugate contains at least ten homing peptides orpeptidomimetics that each selectively homes to tumor blood vessels andtumor cells and that each independently contains the amino acid sequenceSEQ ID NO: 9 or a conservative variant or peptidomimetic thereof. Inanother embodiment, a conjugate of the invention contains at least 100homing peptides or peptidomimetics that each selectively homes to tumorblood vessels and tumor cells and that each independently contains theamino acid sequence SEQ ID NO: 9 or a conservative variant orpeptidomimetic thereof. Any of the above multivalent conjugates of theinvention can include a variety of therapeutic moieties, for example, aphage moiety.

A multivalent conjugate of the invention containing multiple homingmolecules can include, for example, two or more, three or more, five ormore, ten or more, twenty or more, thirty or more, forty or more, fiftyor more, 100 or more, 200 or more, 300 or more, 400 or more, 500 or moreor 100 or more homing molecules. In one embodiment, the homing moleculeshave an identical amino acid sequence. In another embodiment, themultivalent conjugate includes homing molecules having non-identicalamino acid sequences. Moieties useful in a multivalent conjugate of theinvention that incorporates multiple homing molecules include, withoutlimitation, phage, retroviruses, adenoviruses, adeno-associated virusesand other viruses, cells, liposomes, polymeric matrices, non-polymericmatrices or particles such as gold particles, microdevices andnanodevices, and nano-scale semiconductor materials.

A multivalent conjugate of the invention can contain, for example, aliposome or other polymeric matrix linked to at least two homingmolecules that each selectively homes to tumor blood vessels and tumorcells and each specifically binds nucleolin. If desired, the liposome orother polymeric matrix can be linked to at least ten or at least 100 ofsuch homing molecules. Homing molecules useful in such a multivalentconjugate can independently include, for example, the amino acidsequence SEQ ID NO: 9 or a conservative variant or peptidomimetic ofthis sequence. Liposomes consisting, for example, of phospholipids orother lipids, are nontoxic, physiologically acceptable and metabolizablecarriers that are relatively simple to make and administer (Gregoriadis,Liposome Technology, Vol. 1 (CRC Press, Boca Raton, Fla. (1984)). Theliposome or other polymeric matrix additionally can include anothercomponent if desired, such as a therapeutic agent, anti-angiogenic agentor cytotoxic agent.

A conjugate of the invention includes a therapeutic moiety linked to ahoming molecule that selectively homes to tumor blood vessels and tumorcells and that specifically binds nucleolin. As used herein, the term“therapeutic moiety” is used broadly to mean a physical, chemical, orbiological material that can be linked to a homing molecule and thatalters biological activity in a normal or pathologic tissue uponadministration. A therapeutic moiety, therefore, is potentially usefulfor the treatment of disease conditions. A therapeutic moiety can be anynatural or nonnatural material including a biological material, such asa cell or phage; an organic chemical, such as a small molecule; aradionuclide; a nucleic acid molecule or oligonucleotide; a polypeptide;or a peptide or peptidomimetic. Therapeutic moieties useful in theinvention include, without limitation, cancer chemotherapeutic agents;cytotoxic agents; pro-apoptotic agents; and anti-angiogenic agents. Atherapeutic moiety useful in the invention can be expressed on,contained in, or linked to any of the following: phage or other virus,cell, liposome, polymeric or non-polymeric matrix, gold or otherparticle, or a microdevice, nanodevice, or nano-scale semiconductormaterial. These and other materials known in the art can be componentsof the conjugates of the invention.

A therapeutic moiety useful in a conjugate of the invention can be, forexample, an anti-angiogenic agent. As used herein, the term“anti-angiogenic agent” means a molecule that reduces or inhibitsangiogenesis. An anti-angiogenic agent useful in the conjugates andmethods of the invention can be, for example, an inhibitor orneutralizing antibody that reduces the expression or signaling of anangiogenic factor such as vascular endothelial growth factor (VEGF),which is a major inducer of angiogenesis in normal and pathologicalconditions, and is essential in embryonic vasculogenesis. The biologicaleffects of VEGF include stimulation of endothelial cell proliferation,survival, migration and tube formation, and regulation of vascularpermeability. An anti-angiogenic agent also can inhibit anotherangiogenic factor such as a member of the fibroblast growth factor (FGF)family such as FGF-1 (acidic), FGF-2 (basic), FGF-4 or FGF-5 (Slavin etal., Cell Biol. Int. 19:431-444 (1995); Folkman and Shing, J. Biol.Chem. 267:10931-10934 (1992)) or angiopoietin-1, a factor that signalsthrough the endothelial cell-specific Tie2 receptor tyrosine kinase(Davis et al., Cell 87:1161-1169 (1996); and Suri et al., Cell87:1171-1180 (1996)), or the receptor of one of these angiogenicfactors. It is understood that a variety of mechanisms can act toinhibit activity of an angiogenic factor including, without limitation,direct inhibition of receptor binding or of secretion of the angiogenicfactor into the extracellular space, and inhibition of signaling,expression or function of the angiogenic factor.

A variety of anti-angiogenic agents useful in the invention are known inthe art and can be prepared by routine methods. See, for example,Hagedorn and Bikfalvi, Crit. Rev. Oncol. Hematol. 34:89-110 (2000) andKirsch et al., J. Neurooncol. 50:149-163 (2000). Anti-angiogenic agentsinclude, without limitation, small molecules; proteins such asangiogenic factors and receptors, transcription factors, and antibodiesand antigen-binding fragments thereof; peptides and peptidomimetics; andnucleic acid molecules including ribozymes, antisense oligonucleotides,and nucleic acid molecules encoding, for example, dominant negativeangiogenic factors and receptors, transcription factors, and antibodiesand antigen-binding fragments thereof. Exemplary anti-angiogenic agentsuseful in the conjugates and methods of the invention include, yet arenot limited to, angiostatin, endostatin, metastatin and 2ME2 (EntreMed;Rockville, Md.); anti-VEGF antibodies such as Avastin (Genentech; SouthSan Francisco, Calif.); VEGFR-2 inhibitors such as the small moleculesSU5416 and SU6668, (SUGEN; South San Francisco, Calif.); heparin-bindingfragments of fibronectin; modified forms of antithrombin; collagenaseinhibitors; basement membrane turnover inhibitors; angiostatic steroids;platelet factor 4, and fragments and peptides, thereof; thrombospondin,and fragments and peptides thereof; and doxorubicin (O'Reilly et al.,Cell 79:315-328 (1994)); O'Reilly et al., Cell 88: 277-285 (1997);Homandberg et al., Am. J. Path. 120:327-332 (1985); Biochim. Biophys.Acta 874:61-71 (1986); and O'Reilly et al., Science 285:1926-1928(1999)). It is understood that these as well as other anti-angiogenicagents known in the art or that can be prepared by routine methods areencompassed by the term “anti-angiogenic agent” and can be used in thevarious conjugates and methods of the invention.

A therapeutic moiety useful in a conjugate of the invention can be, forexample, a cytotoxic agent. As used herein, the term “cytotoxic agent”refers to any molecule that results in cell death by any mechanism.Exemplary cytotoxic agents useful in a conjugate of the inventionencompass, without limitation, taxanes such as docetaxel; anthracyclinssuch as doxorubicin; alkylating agents; vinca alkaloids;anti-metabolites; platinum agents such as cisplatin or carboplatin;steroids such as methotrexate; antibiotics such as adriamycin;antimicrobial peptides, described herein below; and other cancerchemotherapeutic agents, which are chemical agents that inhibit theproliferation, growth, life-span or metastatic activity of cancer cells.

Taxanes are cytotoxic agents useful in a conjugate of the invention.Useful taxanes include, without limitation, docetaxel (Taxotere; AventisPharmaceuticals, Inc.; Parsippany, N.J.) and paclitaxel (Taxol;Bristol-Myers Squibb; Princeton, N.J.). See, for example, Chan et al.,J. Clin. Oncol. 17:2341-2354 (1999), and Paridaens et al., J. Clin.Oncol. 18:724 (2000).

A cytotoxic agent useful in a conjugate of the invention also can be ananthracyclin such as doxorubicin, idarubicin or daunorubicin.Doxorubicin is a commonly used cancer chemotherapeutic agent (Stewartand Ratain, In: “Cancer: Principles and practice of oncology” 5th ed.,chap. 19 (eds. DeVita, Jr., et al.; J. P. Lippincott 1997); Harris etal., In “Cancer: Principles and practice of oncology,” supra, 1997). Inaddition, doxorubicin has anti-angiogenic activity, which can contributeto its effectiveness in treating cancer (Folkman, supra, 1997; Steiner,In “Angiogenesis: Key principles-Science, technology and medicine,” pp.449-454 (eds. Steiner et al.; Birkhauser Verlag, 1992)).

An alkylating agent such as melphalan or chlorambucil also can be acytotoxic agent useful in a conjugate of the invention. Similarly, vincaalkaloids such as vindesine, vinblastine or vinorelbine; orantimetabolites such as 5-fluorouracil, 5-fluorouridine or a derivativethereof are cytotoxic agents that can be linked to a homing molecule ina conjugate of the invention.

Cytotoxic agents useful in the conjugates of the invention also includeplatinum agents. Such a platinum agent can be, for example, cisplatin orcarboplatin as described, for example, in Crown, Seminars in Oncol.28:28-37 (2001). Other cytotoxic agents useful in a conjugate of theinvention include, without limitation, methotrexate, mitomycin-C,adriamycin, ifosfamide and ansamycins.

A cytotoxic agent also can be, for example, an antimicrobial peptide. Inone embodiment, the invention provides a conjugate in which a homingmolecule that selectively homes to tumor blood vessels and tumor cellsand that specifically binds nucleolin is linked to an antimicrobialpeptide, where the conjugate is selectively internalized by tumor bloodvessels and tumor cells and exhibits a high toxicity to the tumor bloodvessels and tumor cells, and where the antimicrobial peptide has lowmammalian cell toxicity when not linked to the homing molecule. As usedherein, the term “antimicrobial peptide” means a naturally occurring orsynthetic peptide having antimicrobial activity, which is the ability tokill or slow the growth of one or more microbes and which has lowmammalian cell toxicity when not linked to a homing molecule. Anantimicrobial peptide, for example, can kill or slow the growth of oneor more strains of bacteria including a Gram-positive or Gram-negativebacteria, or a fungi or protozoa. Thus, an antimicrobial peptide canhave, for example, bacteriostatic or bacteriocidal activity against, forexample, one or more strains of Escherichia coli, Pseudomonas aeruginosaor Staphylococcus aureus. While not wishing to be bound by thefollowing, an antimicrobial peptide can have biological activity due tothe ability to form ion channels through membrane bilayers as aconsequence of self-aggregation.

An antimicrobial peptide is typically highly basic and can have a linearor cyclic structure. As discussed further below, an antimicrobialpeptide can have an amphipathic α-helical structure (see U.S. Pat. No.5,789,542; Javadpour et al., supra, 1996; Blondelle and Houghten, supra,1992). An antimicrobial peptide also can be, for example, aβ-strand/sheet-forming peptide as described in Mancheno et al., J.Peptide Res. 51:142-148 (1998).

An antimicrobial peptide can be a naturally occurring or syntheticpeptide. Naturally occurring antimicrobial peptides have been isolatedfrom biological sources such as bacteria, insects, amphibians, andmammals and are thought to represent inducible defense proteins that canprotect the host organism from bacterial infection. Naturally occurringantimicrobial peptides include the gramicidins, magainins, mellitins,defensins and cecropins (see, for example, Maloy and Kari, Biopolymers37:105-122 (1995); Alvarez-Bravo et al., Biochem. J. 302:535-538 (1994);Bessalle et al., FEBS 274:151-155 (1990); and Blondelle and Houghten inBristol (Ed.), Annual Reports in Medicinal Chemistry pages 159-168Academic Press, San Diego). As discussed further below, an antimicrobialpeptide also can be an analog of a natural peptide, especially one thatretains or enhances amphipathicity.

An antimicrobial peptide incorporated within a conjugate of theinvention has low mammalian cell toxicity when not linked to a tumorhoming molecule. Mammalian cell toxicity readily can be assessed usingroutine assays. For example, mammalian cell toxicity can be assayed bylysis of human erythrocytes in vitro as described in Javadpour et al.,supra, 1996. An antimicrobial peptide having low mammalian cell toxicityis not lytic to human erythrocytes or requires concentrations of greaterthan 100 μM for lytic activity, preferably concentrations greater than200, 300, 500 or 1000 μM.

In one embodiment, the invention provides a conjugate in which theantimicrobial peptide portion promotes disruption of mitochondrialmembranes when internalized by eukaryotic cells. In particular, such anantimicrobial peptide preferentially disrupts mitochondrial membranes ascompared to eukaryotic membranes. Mitochondrial membranes, likebacterial membranes but in contrast to eukaryotic plasma membranes, havea high content of negatively charged phospholipids. An antimicrobialpeptide can be assayed for activity in disrupting mitochondrialmembranes using, for example, an assay for mitochondrial swelling oranother assay well known in the art. _(D)(KLAKLAK)₂ (SEQ ID NO: 14), forexample, is an antimicrobial peptide which induces marked mitochondrialswelling at a concentration of 10 μM, significantly less than theconcentration required to kill eukaryotic cells.An antimicrobial peptidethat induces significant mitochondrial swelling at, for example, 50 μM,40 μM, 30 μM, 20 μM, 10 μM, or less, is considered a peptide thatpromotes disruption of mitochondrial membranes.

An antimicrobial peptide portion can include, for example, the sequence(KLAKLAK)₂ (SEQ ID NO: 14), (KLAKKLA)₂ (SEQ ID NO: 15), (KAAKKAA)₂ (SEQID NO: 16), or (KLGKKLG)₃ (SEQ ID NO: 17), and, in one embodiment,includes the sequence _(D)(KLAKLAK)₂(SEQ. ID NO: 14. A conjugate of theinvention, which contains a homing molecule that selectively homes totumor blood vessels and tumor cells linked to an antimicrobial peptide,can have, for example, the sequence KDEPQRRSARLSAKPAPPKPEPKPKKAPAKK-GG-_(D)(KLAKLAK)₂ (SEQ ID NO: 21).

Antimicrobial peptides generally have random coil conformations indilute aqueous solutions, yet high levels of helicity can be induced byhelix-promoting solvents and amphipathic media such as micelles,synthetic bilayers or cell membranes. α-Helical structures are wellknown in the art, with an ideal α-helix characterized by having 3.6residues per turn and a translation of 1.5 Å per residue (5.4 Å perturn; see Creighton, Proteins: Structures and Molecular Properties W.HFreeman, New York (1984)). In an amphipathic α-helical structure, polarand non-polar amino acid residues are aligned into an amphipathic helix,which is an α-helix in which the hydrophobic amino acid residues arepredominantly on one face, with hydrophilic residues predominantly onthe opposite face when the peptide is viewed along the helical axis.

Antimicrobial peptides of widely varying sequence have been isolated,sharing an amphipathic α-helical structure as a common feature (Saberwalet al., Biochim. Biophys. Acta 1197:109-131 (1994)). Analogs of nativepeptides with amino acid substitutions predicted to enhanceamphipathicity and helicity typically have increased antimicrobialactivity. In general, analogs with increased antimicrobial activity alsohave increased cytotoxicity against mammalian cells (Maloy et al.,Biopolymers 37:105-122 (1995)). Synthetic, antimicrobial peptides havingan amphipathic α-helical structure are known in the art, for example, asdescribed in U.S. Pat. No. 5,789,542 to McLaughlin and Becker.

Effective cytotoxic agents include those that target DNA, for example,alkylating agents, agents that intercalate into DNA, and agents whichresult in double-stranded DNA breaks. Exemplary DNA-targeted drugsinclude, without limitation, cyclophosphamide, melphalan, mitomycin C,bizelesin, cisplatin, doxorubicin, etoposide, mitoxantrone, SN-38,Et-743, actinomycin D, bleomycin, TLK286 and SGN-15 (Hurley, supra,2002).

A therapeutic moiety for treatment of breast cancer or anotherhormonally-dependent cancer also can be an agent that antagonizes theeffect of estrogen, such as a selective estrogen receptor modulator oran anti-estrogen. The selective estrogen receptor modulator, tamoxifen,is a cancer chemotherapeutic agent that can be used in a conjugate ofthe invention for treatment of breast cancer (Fisher et al., J. Natl.Cancer Instit. 90:1371-1388 (1998)).

A therapeutic moiety useful in a conjugate of the invention also can bean antibody such as a humanized monoclonal antibody. As an example, theanti-epidermal growth factor receptor 2 (HER2) antibody, trastuzumab(Herceptin; Genentech, South San Francisco, Calif.) is a therapeuticagent useful in a conjugate of the invention for treating HER2/neuoverexpressing breast cancers (Burris et al., supra, 2001; White et al.,Annu. Rev. Med. 52:125-141 (2001)).

It is understood by one skilled in the art of medicinal oncology thatthese and other agents are useful therapeutic moieties, which can beused separately or together in the conjugates and methods of theinvention. It further is understood that a conjugate of the inventioncan contain one or more of such therapeutic moieties and that additionalcomponents can be included as part of the conjugate, if desired. As anexample, in some cases it can be desirable to utilize an oligopeptidespacer between the homing molecule and the therapeutic agent(Fitzpatrick and Garnett, Anticancer Drug Des. 10:1-9 (1995)).

A conjugate of the invention also can include a detectable label. Asused herein, the term “detectable label” refers to any molecule whichcan be administered in vivo and subsequently detected. Exemplarydetectable labels useful in the conjugates and methods of the inventioninclude radiolabels and fluorescent molecules. Exemplary radionuclidesinclude indium-111, technetium-99, carbon-11, and carbon-13. Fluorescentmolecules include, without limitation, fluorescein, allophycocyanin,phycoerythrin, rhodamine, and Texas red.

The present invention also provides methods of directing a therapeuticmoiety to tumor blood vessels and tumor cells in a subject byadministering to the subject a conjugate which contains a therapeuticmoiety linked to a homing molecule that selectively homes to tumor bloodvessels and tumor cells and that specifically binds nucleolin, therebydirecting the therapeutic moiety to tumor blood vessels and tumor cells.In one embodiment, the homing molecule is not an antibody orantigen-binding fragment thereof. In other embodiments, the peptide orpeptidomimetic portion of the conjugate has a length of at most 200residues, or a length of at most 50 residues.

A variety of homing molecules are useful in the methods of the inventionincluding homing peptides and peptidomimetics. A method of directing atherapeutic moiety to tumor blood vessels and tumor cells in a subjectcan be practiced, for example, using a homing peptide or peptidomimeticthat contains the amino acid sequence SEQ ID NO: 9, or a conservativevariant or peptidomimetic of this sequence. In one embodiment, such ahoming peptide or peptidomimetic includes the amino acid sequence SEQ IDNO: 9, or a peptidomimetic thereof. A method of directing a therapeuticmoiety to tumor blood vessels and tumor cells in a subject also can bepracticed, for example, with a homing peptide or peptidomimetic whichcontains the amino acid sequence SEQ ID NO: 11, or a conservativevariant or peptidomimetic of this sequence. In one embodiment, themethod is practiced with a conjugate containing a homing peptide orpeptidomimetic that includes the amino acid sequence SEQ ID NO: 11 or apeptidomimetic thereof.

A variety of therapeutic moieties can be directed to tumor blood vesselsand tumor cells in a subject according to a method of the invention.Such moieties encompass, without limitation, anti-angiogenic agents andcytotoxic agents, including cytotoxic agents that target aDNA-associated process such as alkylating agents, anti-tumor antibioticsand sequence-selective cytotoxic agents. In particular embodiments, amethod of the invention relies on one of the following cytotoxic agentsthat target a DNA-associated process: cyclophosphamide, melphalan,mitomycin C, bizelesin, cisplatin, doxorubicin, etoposide, mitoxantrone,SN-38, Et-743, actinomycin D, bleomycin or TLK286.

The present invention also provides a method of imaging tumors and tumorvasculature in a subject by administering to the subject a conjugatecontaining a detectable label linked to a homing molecule thatselectively homes to tumor blood vessels and tumor cells and thatspecifically binds nucleolin; and detecting the conjugate, therebyimaging tumors and tumor vasculature. A homing molecule useful in animaging method of the invention can be, for example, a homing peptide orpeptidomimetic such as a homing peptide or peptidomimetic that containsthe amino acid sequence SEQ ID NO: 9 or a conservative variant orpeptidomimetic of this sequence. Any of a variety of detectable labelsare useful in the imaging methods of the invention, includingfluorescent labels and radionuclides such as indium-111, technetium-99,carbon-11, and carbon-13.

The methods of the invention for imaging tumors and tumor vasculaturecan be useful for detecting the presence of blood vessels associatedwith a variety of tumors. Following administration of a conjugate of theinvention containing a detectable label, tumor blood vessels arevisualized. If the image is positive for the presence of such tumorvessels, the tumor can be evaluated for size and quantity of vascularinfiltration. These results provide valuable information to theclinician with regard to the stage of development of the cancer and thepresence or probability of metastasis.

In a method of imaging tumors and tumor vasculature, the conjugateadministered contains a detectable label that allows detection orvisualization of tumor blood vessels and tumor cells, for example, ofleukemias or breast cancers. For in vivo diagnostic imaging of suchcancers, a homing molecule is linked to a detectable label that, uponadministration to the subject, is detectable external to the subject.Such a detectable label can be, for example, a gamma ray emittingradionuclide such as indium-113, indium-115 or technetium-99; followingadministration to a subject, the conjugate can be visualized using asolid scintillation detector.

The present invention also provides a method of reducing the number oftumor blood vessels in a subject by administering to the subject aconjugate which contains a cytotoxic agent linked to a homing moleculethat selectively homes to tumor blood vessels and tumor cells and thatspecifically binds nucleolin, thereby reducing the number of tumor bloodvessels in the subject. The peptide or peptidomimetic portion of theconjugate can have, for example, a length of at most 200 residues, or alength of at most 50 residues. In one embodiment, a method of theinvention is practiced with a conjugate containing a homing peptide orpeptidomimetic. In a further embodiment, a method of the invention ispracticed with a conjugate containing a homing peptide or peptidomimeticthat includes the amino acid sequence SEQ ID NO: 9, or a conservativevariant or peptidomimetic of this sequence. Any of the therapeuticmoieties described above, such as anti-angiogenic agents, cytotoxicagents and cytotoxic agents that target a DNA-associated process, aswell as additional moieties disclosed herein or known in the art, can beused to reduce the number of tumor blood vessels according to a methodof the invention.

Also provided herein is a method of treating cancer in a subject byadministering to the subject a conjugate which contains a therapeuticmoiety linked to a homing molecule that selectively homes to tumor bloodvessels and tumor cells and that specifically binds nucleolin. Inparticular embodiments, the peptide or peptidomimetic portion of theconjugate has a length of at most 200 residues, or a length of at most50 residues. In other embodiments, a method of the invention ispracticed with a conjugate containing a homing peptide or peptidomimeticsuch as a homing peptide or peptidomimetic that includes the amino acidsequence SEQ ID NO: 9, or a conservative variant or peptidomimetic ofthis sequence. It is understood that, in a method of the invention fortreating cancer in a subject, any of a variety of therapeutic moietiescan be useful, including but not limited to, anti-angiogenic agents;cytotoxic agents; and cyclophosphamide, melphalan, mitomycin C,bizelesin, cisplatin, doxorubicin, etoposide, mitoxantrone, SN-38,Et-743, actinomycin D, bleomycin, TLK286 and other cytotoxic agents thattarget a DNA-associated process.

It is understood that a variety of routes of administration are usefulin the methods of the invention. Such routes encompass systemic andlocal administration and include, without limitation, oraladministration, intravenous injection, intraperitoneal injection,intramuscular injection, subcutaneous injection, transdermal diffusionor electrophoresis, local injection; extended release delivery devices,including locally implanted extended release devices such as bioerodibleor reservoir-based implants.

The present invention further provides a method of isolating progenitorcells from a heterogeneous mixture of cells by contacting theheterogenous mixture of cells with a homing molecule that selectivelyhomes to tumor blood vessels and tumor cells and specifically bindsnucleolin under conditions suitable for specific binding of the homingmolecule to the progenitor cells; and separating cells that bind thehoming molecule from non-binding cells, thereby isolating progenitorcells from the heterogenous mixture of cells. The heterogeneous mixtureof cells can be, for example, primary tissue such as primary bonemarrow.

In one embodiment, the homing molecule used to isolate progenitor cellsaccording to a method of the invention is a homing peptide orpeptidomimetic. In a further embodiment, the method is practiced with ahoming peptide or peptidomimetic containing the amino acid sequence SEQID NO: 9 or a conservative variant or peptidomimetic thereof. In anotherembodiment, the method is practiced with a homing peptide orpeptidomimetic containing the amino acid sequence SEQ ID NO: 11 or aconservative variant or peptidomimetic thereof. Homing peptides andpeptidomimetics useful in isolating progenitor cells can have a varietyof lengths, including, without limitation, a length of less than 85residues, a length of less than 50 residues, or a length of less than 35residues.

A method of the invention for isolating progenitor cells can bepracticed, if desired, with a homing molecule attached to a support. Amethod of the invention also can be practiced, for example, with ahoming molecule linked to a fluorescent label. In one embodiment, theseparation step includes fluorescence activated cell sorting (FACS). Infurther embodiments, progenitor cells are isolated using a homingpeptide or peptidomimetic containing the amino acid sequence SEQ ID NO:9 or the amino acid sequence SEQ ID NO: 11, or a conservative variant orpeptidomimetic of one of these sequences, linked to a fluorescent label.

The following examples are intended to illustrate but not limit thepresent invention.

EXAMPLE I In Vivo Homing of a Fragment of HMGN2

This example demonstrates that an amino-terminal fragment of HMGN2selectively homes to tumor blood vessels and tumor cells in vivo.

Hematopoietic and endothelial precursors originate from a commonprecursor, hemangioblasts. Based on the shared phenotypiccharacteristics of hematopoietic and endothelial precursors, a phagescreening procedure was devised to select cDNA clones that bind anepitope shared by both primitive bone marrow cells and angiogenicendothelial cells. The screening procedure included an ex vivo primaryselection on lineage-depleted murine bone marrow cells to select forbinding to endothelial progenitor cells and a further selection forhoming to HL-60 xenograft tumors in vivo.

After two rounds of pre-selection on lineage-depleted murine bone marrowcells, the resulting phage pool was injected into the tail vein of nudemice bearing HL-60 tumors. After 10 minutes of circulation, the micewere perfused through the heart, and the phage rescued from variousorgans, amplified, and used for subsequent rounds of selection. As shownin FIG. 1, the selected phage pool exhibited 20-fold enrichment fortumor homing relative to the unselected library after two rounds of invivo selection. Sequencing analysis showed that the predominant cDNA inthe selected pool was a 270-bp clone (SEQ ID NO: 5) that contained anopen reading frame encoding the first 73 amino-terminal residues ofhuman HMGN2 as well as 51 bp of 5′ non-coding sequence (FIG. 2A). Asfurther shown in FIG. 2A, additional HMGN2 clones (SEQ ID NOS: 1 through4) also were isolated from the phage pool; all the HMGN2 clones shared acommon sequence corresponding to exons 3 and 4 in the HMGN2 sequence(see FIG. 2A).

An independent screening strategy also resulted in isolation of the sameHMGN2 cDNA clone, SEQ ID NO: 5. In this second strategy, an initialpre-selection was performed by assaying for in vitro binding to murinebone marrow cells positive for the progenitor cell marker, CD34,followed by in vivo selection in nude mice bearing MDA-MB-435 humanbreast cancer xenografts.

Purified phage bearing the HMGN2 fragment SEQ ID NO: 5 homed to HL-60tumors in vivo to about the same extent as the selected pool. Thepurified phage displaying SEQ ID NO: 5 also accumulated in the kidneysif the number of injected phage was small (1×10⁹ plaque forming units(pfu)). In contrast, homing to tumors was observed using either 1×10⁹pfu or 1×10¹² pfu injected phage.

As expected, phage bearing the HMGN2 fragment SEQ ID NO: 5 alsoexhibited a preference for binding tumor cells in vitro. In particular,about 1000 times more SEQ ID NO: 5-displaying phage than non-recombinantT7 phage bound to cultured HL-60 or MDA-MB-435 cells in vitro.Similarly, SEQ ID NO: 5-displaying also bound cell suspensions preparedfrom HL-60 tumors with a 1000-fold specificity relative to control T7phage. These results indicate that the HMGN2 fragment SEQ ID NO: 5 issufficient for selective homing to tumors of different types.

Cell lines were maintained and xenograft tumors were established asfollows. Human myeloid leukemia HL-60 (ATTC) and MDA-MB-435 human breastcarcinoma cell lines were grown in RPMI-1640 media supplemented with 10%fetal bovine serum (Arap et al., Science, 279:377-380 (1998)). Toestablish xenograft tumors, 2 to 3-month old nude mice (Harlan SpragueDawley; San Diego, Calif.) were injected subcutaneously with 10⁶exponentially growing tumor cells in 200 μl culture media. HL-60 andMDA-MB-435 xenograft bearing animals were used in experiments within 3-5weeks or 8-10 weeks, respectively, of the time of the injection.

cDNA synthesis, cloning, and phage packaging and amplification wereperformed essentially as follows. Phage cDNA libraries were preparedusing mRNA purified from normal (human bone marrow, brain or mouseembryo) or malignant (liver, lung, breast, and colon carcinoma) tissues(BD Biosciences Clontech; Palo Alto, Calif.) and from mouse spleen andbone marrow (Oligotex Direct mRNA kit; Qiagen; Valencia, Calif.). cDNAsynthesis was performed with random primers; cDNAs were cloned into theT7Select 10-3b vector; and phage were packaged and amplified accordingto the manufacturer's instructions (Novagen; Madison, Wis.). cDNAlibraries were pooled for the phage screening.

Murine bone marrow progenitor cells were isolated and depleted of cellsbearing several lineage-specific markers essentially as follows. Mousebone marrow was obtained by flushing femoral and tibial bones with 3 mlcold media (DMEM supplemented with 10% FBS). Bone marrow subsequentlywas depleted of cells expressing common lineage-specific markers byusing the StemSep Murine Kit (StemCell Technologies; Vancouver, Canada)with antibodies recognizing the following antigens coupled toparamagnetic beads: mouse CD5 (clone Ly-1); myeloid differentiationantigen (Gr-1); CD45R (B220); erythroid cells (TER119); CD11b (Mac-1);and neutrophils (7-4; StemCell Technologies). After depletion of cellsbearing lineage-specific markers, the remaining megakaryocytes wereremoved by filtering through a 30 μm nylon mesh filter (MiltenyiBiotech; Auburn, Calif.).

Ex vivo and in vivo selections were performed as described (Rajotte etal., Journal of Clinical Investigation 102:430-437 (1998); Hoffman, etal., in Phage Display: A Practical Approach, eds. Clarkson, T. & Lowman,H., Oxford University Press, Oxford, UK (2002), In press; Laakkonen etal., Nature Medicine In revision, 28-30 (2002)). Briefly, 1×10⁹ pfu ofphage library were incubated with target cells overnight at 4° C. Afterunbound phage were removed by extensive washing, phage bound to cellswere rescued, amplified, and used for the subsequent round of selection.After two rounds of in vitro selection, the phage pool was subjected toin vivo selection by injecting the pool (1×10⁹ pfu) into the tail veinof a nude mouse bearing an HL-60 xenograft tumor. After two rounds of invivo selection, 96 phage clones were selected from the pool oftumor-homing phage. Protein-encoding inserts were sequenced by standardmethods.

EXAMPLE II Delineation of the HMGN2 Tumor Cell-Binding Domain

This example describes localization of the HMGN2 domain responsible fortumor cell binding and in vivo homing activity.

A. Identification of an HMGN2-Derived Peptide Sequence Sufficient forTumor Cell Binding and in Vivo Homing Activity.

Phage displaying a set of sequences corresponding to fragments of theamino-terminal portion of HMGN2 (SEQ ID NO: 5) were constructed tolocalize the region responsible for cell binding and in vivo homing.Fragments were designed to follow the exon/intron boundaries of theHMGN2 gene. Inserts encoding the indicated fragments were amplified fromthe full length HMGN2 phage clone by PCR, purified, digested anddirectionally cloned into the T7 415-1 and 10-3 vectors. Phage werepackaged, amplified and sequenced according to the manufacturer'sinstructions. Phage preparations were then tested for binding to primarycells obtained from HL-60 xenograft tumors. After a 1 hour incubation at4° C., cells were washed, and bound phage quantified. As shown in FIG.2B, when phage bearing fragments SEQ ID NO: 7, 8, 9 and 10 were testedfor tumor binding activity as compared to non-recombinant phage, onlythe 31-amino acid fragment encoded by exons 3 and 4 (SEQ ID NO: 9),which corresponds to the nucleosomal binding domain of HMGN2,demonstrated substantial tumor cell binding (FIG. 2B). Furthermore,phage displaying the N-terminal portion of the active fragment SEQ IDNO: 9, which corresponds to exon 3 of the HMGN2 gene, were prepared.These phage expressing the sequence PQRRSARLSA (SEQ ID NO: 11) bound totumor cells 90-fold more than non-recombinant phage.

The phage binding assay was performed essentially as follows. Attachmentof phage to cells was quantified by incubating 1×10⁸ pfu of phagedisplaying SEQ ID NO: 9 for 60 minutes with 1×10⁶ HL-60 cells at 4° C.in Tris-buffered saline with 1 mM Ca⁺² and 1 mM MgCl₂. Bound phage wererescued after four washes with phosphate-buffered saline by adding 1 mlof bacteria for 7 minutes at room temperature. Bound phage werequantified by plating and counting pfu.

B. Specificity of Active Peptide Binding

Binding of SEQ ID NO: 9-displaying phage to tumor cells was inhibited byfree SEQ ID NO: 9 peptide in a dose dependent fashion. Completeinhibition was achieved with 100 μM free peptide SEQ ID NO: 9,indicating that phage binding was specific. In contrast, backgroundbinding of non-recombinant phage was unaffected by free peptide.Specificity of cell binding by peptide SEQ ID NO: 9 was furtherconfirmed by comparing HL-60 cell binding of phage expressing the HMGN2exon 3 sequence PQRRSARLSA (SEQ ID NO: 11) to binding with phageexpressing the homologous HMGN1 exon 3 sequence PKRRSARLSA (SEQ ID NO:12). The HMGN1 phage expressing SEQ ID NO: 12 bound 90% less than theHMGN2 phage expressing SEQ ID NO: 11, indicating that the single aminoacid change from glutamine to lysine substantially alters cell bindingspecificity of the homing fragment.

These results indicate that binding of SEQ ID NO: 9-displaying phage isdue to the specific binding activity of SEQ ID NO: 9.

EXAMPLE III Tissue and Sub-Cellular Localization of HMGN2 Peptide SEQ IDNO: 9

This example demonstrates that peptide SEQ ID NO: 9 accumulates in tumorcells and cells lining the blood vessels upon intravenousadministration.

A. Histological Analysis of HMGN2 Peptide Homing

To study peptide localization, fluorescein-conjugated SEQ ID NO: 9 orARALPSQRSR (SEQ ID NO: 13) was injected into the tail vein of micebearing HL-60 or MDA-MB-435 xenografts. Peptide injection was followed10 minutes later by injection of biotinylated tomato lectin, a marker ofthe vasculature. After another five minutes, mice were perfused throughthe heart with fixative solution, and the organs dissected, sectionedand stained with streptavidin-AlexaFluor 594. Slides werecounter-stained with DAPI and examined under an inverted fluorescentmicroscope. As shown in FIG. 3A, strong fluorescence was present intumor tissue, whereas little or no specific fluorescence was detected innormal brain, liver or spleen. See, for example, FIG. 3B, which showsimmunofluorescence of brain tissue. HMGN2 peptide SEQ ID NO: 9 also waspresent in a small population of cells in the skin and the gut, whichwere not associated with the vasculature and which may representprogenitor cells (see FIGS. 3C and D). In addition, diffuse fluorescenceaccumulated in proximal tubules of kidneys following injection withpeptide SEQ ID NO: 9 or fluorescein-labeled scrambled exon 3 peptide(ARALPSQRSR; SEQ ID NO: 13), indicating that kidney staining was due tonon-specific uptake of peptide or fluorescein from the glomerularfiltrate. The control peptide ARALPSQRSR (SEQ ID NO: 13) was essentiallyundetectable in other tissues, including the HL-60 tumors, as shown inFIG. 3E.

Within the HL-60 leukemia tumor tissue, SEQ ID NO: 9 localized to tumorcells and cells lining tumor blood vessels. As shown in FIGS. 3A andG-J, fluorescence predominantly localized to nuclei, with most of thetumor cells containing fluorescence positioned close to blood vessels.Fluorescein-conjugated peptide SEQ ID NO: 9 also accumulated inendothelial cells and tumor cells in mice expressing MDA-MB-435 breastcancer xenografts. In some microscopic fields, SEQ ID NO: 9 fluorescenceessentially was limited to the endothelial cells, clearly illustratingthe expression of peptide SEQ ID NO: 9 within endothelial cells andtheir nuclei (see FIG. 3F). Similar tumor localization was obtained whenpeptide SEQ ID NO: 9 was coupled to another fluorescent molecule,rhodamine. In sum, these results indicate that peptide SEQ ID NO: 9selectively homes to tumors and tumor endothelial cells as well as aminor population of progenitor cell-like bone marrow cells and a smallpopulation of cells in normal skin and gut.

Peptides were synthesized with an automated peptide synthesizer by usingstandard solid-phase Fmoc chemistry (Atherton and Sheppard, Solid-PhasePeptide Synthesis, IRL, Oxford (1989)). Peptides were labeled withfluorescein via an amino-hexanoic acid spacer during peptide synthesisas described in Wender et al., Proc. Nat. Acad. Sci. USA 97: 13003-13008(2000). The concentration of unlabeled peptide was determined byweighing and from absorbance at 230 nm (Ehresmann et al., AnalyticalBiochemistry 54:454-463 (1973)).

Histological analyses were performed as follows. Tissue distribution ofhoming ligands was examined by intravenously injectingfluorescein-coupled peptides (100 μl of 1 mM solution) into the tailvein of anesthesized mice bearing HL-60 xenografts prepared as describedabove. Blood vessels were visualized by intravenously injecting 200 μlof 0.5 μg/μl biotin-conjugated tomato lectin (Vector Laboratories;Burlingame, Calif.). Peptide was injected first, followed by lectin, andthe injected materials were allowed to circulate for five minutes. Themouse, which remained anesthesized throughout the experiment, wasperfused subsequently through the heart with 4% paraformaldehyde. Organswere removed and frozen in O.C.T. embedding medium (Tissue-Tek; Elkhart,Ind.). Biotin-conjugated lectin was detected with streptavidin-Alexa 594(Molecular Probes; Eugene, Oreg.); the slides were mounted withVectashield-DAPI (Vector Laboratories; Burlingame, Calif.), and examinedunder an inverted fluorescent microscope.

B. Peptide SEQ ID NO: 9 Binds Cells in Human Bone Marrow with ProgenitorCell-Like Characteristics

Fluorescein-labeled SEQ ID NO: 9 or control peptide SEQ ID NO: 13 (2 μM)were incubated with gradient-depleted bone marrow cells for one hour at4° C. and analyzed in a flow cytometer. As shown in FIG. 4, peptide SEQID NO: 9 specifically bound to a small cell population in human bonemarrow, representing about 0.3-0.8% of mononuclear cells. The SEQ ID NO:9-positive cells were the size of lymphocytes, agranular, CD45-positiveand mostly CD34-negative, a marker profile similar to that of thelineage-depleted murine bone marrow cells used in the in vitro phageselection. These results indicate that peptide SEQ ID NO: 9 binds aminor population of progenitor cell-like bone marrow cells.

Flow cytometry was performed as follows. Human bone marrow specimensrepresented excess material from samples collected for diagnosticpurposes from adults with hematological malignancies following informedconsent. A total of 2 ml of bone marrow was aspirated from the posterioriliac crest and stored in a citrate anticoagulant. Mononuclear cellswere isolated by gradient centrifugation (Ficoll-Paque PLUS; AmershamPharmacia Biotech; Uppsala, Sweden) and incubated in RPMI-1640 mediasupplemented with 10% FBS for two hours at 37° C. Cells subsequentlywere transferred to 4° C. and incubated with 1-2 μMfluorescein-conjugated peptide for 45 minutes before staining withPerCP- or PE-conjugated CD34 and CD45 antibodies (Beckton DickinsonBiosciences; San Jose, Calif.) for 30 minutes. Samples were analyzedwith either a FACSCalibur or LSR flow cytometer (Beckton DickinsonBiosciences); 100,000 events were collected.

C. Rapid Nuclear Uptake of Peptide SEQ ID NO: 9 in Tumor Cells in Vitro

Cellular uptake and nuclear translocation of peptide SEQ ID NO: 9 wereobserved in cultured HL-60 cells and MDA-MB-435 cells in vitro. HL-60 orMDA-MB-435 cells were incubated with 1 μM fluorescein-labeled SEQ ID NO:9 or control peptide SEQ ID NO: 13 at 370. After 30 minutes, cells werewashed, fixed, stained with nuclear counter-stain (DAPI; blue), andimaged under a confocal or epifluorescent microscope. As shown in FIG.5, peptide SEQ ID NO: 9 rapidly localized to the nucleus. The uptake ofpeptide SEQ ID NO: 9 did not occur efficiently at 4° C., indicatingenergy dependence. Furthermore, while the D-amino acid form of peptideSEQ ID NO: 9 also was internalized by MDA-MB-435 cells, albeit moreslowly than the L-form, the D-form did not accumulate in the nucleus.These properties were reminiscent of the cellular uptake and nucleartranslocation of highly basic peptides from Tat protein and certainhomeobox proteins (Lindgren et al., Trends in Pharmacological Sciences21:99-103 (2000); Schwarze et al., Science 285:1569-1572 (1999) andGallouzi and Steitz, Science 294:1895-1901 (2001)).

In sum, these results demonstrate that peptide SEQ ID NO: 9 localizes tothe nuclei of tumor cells upon internalization and further indicate thatpeptide SEQ ID NO: 9 and molecules that bind the same receptor can beuseful for selectively targeting anti-cancer drugs to tumor vasculature.

EXAMPLE IV Identification of Homing Molecules that Target the ReceptorBound by SEQ ID NO: 9

This example describes a routine binding competition assay foridentification of homing molecules that bind the receptor bound by SEQID NO: 9.

Phage binding to cells is quantified by incubating 1×10⁸ plaque formingunits (pfu) of phage displaying SEQ ID NO: 9 for 60 minutes with 1×10⁶HL-60 cells at 4° C. in Tris buffered saline containing 1 mM Ca⁺² andMg⁺². After four washes with phosphate-buffered saline, bound phage arerescued by addition of 1 ml bacteria for 7 minutes at room temperature.Bound phage are quantified by plating and counting pfu.

Inhibition of phage binding is determined by adding varyingconcentrations of a test molecule to the mixture of phage and HL-60cells. Any significant inhibition of phage binding is an indication thatthe test molecule specifically binds to the same receptor as SEQ ID NO:9 and is a homing molecule that selectively homes to tumor blood vesselsand tumor cells.

EXAMPLE V Isolation of Progenitor Cells

This example describes a procedure for purification of progenitor cellsfrom bone marrow or another tissue source. Tumor cells or tumor bloodvessels can be similarly purified using the appropriate tissue source.

Organs are removed from mice, rinsed in PBS, minced into approximately 1mm squares, and digested in 10 ml collagenase A (1 mg/ml; SIGMA), DNaseI (25 μg/ml; SIGMA), and Dispase II (2.4 U/ml; Roche) at 37° C. for 1.5hours with continuous rotation. The cell suspension is filtered through50 μm nylon mesh, centrifuged at 1000×g for five minutes, and washedonce in PBS with 2% FCS and 5% rat serum. Cells are suspended andincubated with fluorescein-labeled peptide SEQ ID NO: 9 at 50 μg/ml, andmouse monoclonal anti-fluorescein antibody (Molecular Probes) for 15minutes. The cell suspension is then centrifuged at 1000×g for fiveminutes. After resuspending the cell pellet in 90 μl buffer and 10 μl ofanti-mouse IgG microbeads (Miltenyi Biotec), the mixture is rotated inthe cold room for 15 minutes. After two washes with buffer, cells areresuspended in 0.5 ml buffer and applied to a MACS separation column onthe MACS MultiStand. The column is washed with 1 ml of degassed buffer,and the cells are flushed out with the column plunger. The purity of theSEQ ID NO: 9-reactive cells is examined by incubating cells withfluorescein-coupled peptide SEQ ID NO: 9 at 25 μg/ml), and analyzingcells under a fluorescent microscope.

These results indicate that SEQ ID NO: 9-reactive cells can be purifiedby routine methods.

All journal article, reference and patent citations provided above, inparentheses or otherwise, whether previously stated or not, areincorporated herein by reference in their entirety.

Although the invention has been described with reference to the examplesprovided above, it should be understood that various modifications canbe made without departing from the spirit of the invention. Accordingly,the invention is limited only by the claims.

EXAMPLE VI Identification of the HMGN2 Target Molecule

This example demonstrates that cell surface nucleolin is a novel markerfor tumor endothelium.

A. Affinity Chromatography Demonstrates that Nucleolin Binds Peptide SEQID NO: 9

As indicated above, cultured tumor cells, such as the human breastcarcinoma cell line MDA-MB-435, bind the HMGN2-derived peptide, SEQ IDNO: 9, in vitro. As shown in FIG. 6A, affinity chromatography ofMDA-MB-435 cell extracts revealed a major band at a molecular weight of110 kDa and several bands in the 20 kDa range that bound to peptide SEQID NO: 9 but not to control peptide. Mass spectrometric analysisindicated that the 110 kDa band represents nucleolin. The 20 kDa rangebands were identified as various histones.

Affinity chromatography with peptide SEQ ID NO: 9 and MDA-MB-435detergent extracts was performed essentially as described in Christianet al., J. Biol. CHem. 276:48588-48595 (2001). Briefly, 6×10⁸ MDA-MB-435cells grown in RPMI 1640 medium with 10% fetal calf serum were pelletedand lysed in 60 ml of RIPA buffer (1% Triton X-100, 0.5% deoxycholicacid, 0.1% SDS, 10 mM Tris-HCl pH 7.6, 150 mM NaCl, and 1% ProteaseInhibitor Cocktail for Mammalian Cells; SIGMA). The lysate was incubatedwith 20 ml of peptide SEQ ID NO: 9 or control peptide(APKDKPAAVKERKKPAPKPRPQELRSKKAKPAPAS; SEQ ID NO:20) affinity matrix (2mg of peptide covalently coupled to 1 ml of Affigel 10). Matrix beadswere washed three times with IP-wash buffer (0.025% Triton X-100, 50 mMTris-HCl pH 8.4, 150 mM NaCl, 1 mM CaCl₂, and 0.02% azide), two timeswith 25 mM Tris-HCl pH 8.4/250 mM NaCl, and eluted with 30 μl of SDS gelsample buffer. Affinity-purified proteins were reduced with 50 mM DTTbefore being separated on an 8-20% polyacrylamide gel and visualized byColloidal Blue staining (Invitrogen; Carlsbad, Calif.). Bands thatappeared in the peptide SEQ ID NO: 9 eluate, but not in control eluate,were cut out, digested with trypsin and analyzed by mass spectroscopyusing matrix-assisted laser desorption ionization-time of flightanalysis (Voyager DE-PRO, Applied Biosystems; Foster City, Calif.).Peptide samples were prepared using an alpha-cyano-4-hydroxycinnamicacid (HCCA)/nitrocellulose matrix.

Identification of the 110-kDa protein as nucleolin was confirmed byimmunoblotting. A monoclonal antibody against nucleolin revealed a major110-kDa band and a faint lower molecular size band in the peptide SEQ IDNO: 9-bound material (see FIG. 6B). These bands were not present ineluates from the control matrix. In the unfractionated sample(“extract”), anti-nucleolin antibody recognizes full-length nucleolin at110 kDa, along with several faster-migrating bands, including one at 75kDa. The faint bands in the material obtained by purification on SEQ IDNO: 9 matrix aligned with several of the lower molecular size bandsdetected by anti-nucleolin antibody in whole cell extracts, which arelikely nucleolin fragments. These results show that the HMGN2-derivedpeptides such as peptide SEQ ID NO: 9 specifically bind nucleolin.

Immunoblot analysis was performed as follows. Cell extracts oraffinity-purified material was separated by SDS-PAGE and transferred onnitrocellulose membrane for one hour at 100V. The membrane was blockedovernight at 4° C. in 5% milk powder in TBS-T (140 mM NaCl, 10 mMTris-HCl pH 7.4, 0.05% Tween) and incubated with 10 mg/ml mousemonoclonal IgG₁ MS-3 anti-nucleolin antibody (Santa Cruz Biotechnology;Santa Cruz, Calif.) in TBS-T for one hour at room temperature. Afterextensive washing, the membrane was incubated with peroxidase coupledrabbit anti-mouse antibody; bound antibody was detected with enhancedchemiluminescence (ECL; Amersham) and exposure to Biomax MR (Kodak;Rochester, N.Y.).

B. Nucleolin is Expressed at the Cell Surface

Nucleolin was originally described as a nuclear protein, although thisprotein has also been observed at the cell surface (Sinclair andO'Brien, J. Biol. Chem. 277:2876-2885 (2002); and Said et al., J. Biol.Chem. 277:37492-37502 (2002)). To determine if the SEQ ID NO: 9-bindingnucleolin in MDA-MB-435 cells is expressed at the cell surface,MDA-MB-435 cells were biotinylated with a cell-impermeable biotinreagent, and the resulting cell extracts assayed for the ability to bindSEQ ID NO: 9. As shown in FIG. 7A, affinity purification on immobilizedpeptide SEQ ID NO: 9 identified two streptavidin-reactive bands at110-kDa and 75-kDa that specifically bound to peptide SEQ ID NO: 9. Themolecular weights of these bands were similar to the SEQ ID NO:9-binding nucleolin bands detected with anti-nucleolin antibody. Asexpected, histones, which bound the SEQ ID NO: 9 peptide matrix in acell extract, were not biotin-labeled in intact cells.

Cell surface biotinylation experiments were performed essentially asfollows. MDA-MB-435 cells (5×10⁶ cells) were washed three times withcold phosphate-buffered saline on a cell culture plate and incubatedwith biotinylation buffer (20 mM HEPES, pH 7.45, 5 mM KCl, 130 mM NaCl,0.8 mM MgCl₂, 1 mM CaCl₂, 0.5 mg/ml EZ link Sulfo-NHS-Biotin; Pierce)for one hour at 4° C. After removal of reagent, cells were washed threetimes with wash buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1 mM MgCl₂, 1mM CaCl₂) and lysed in 1% Triton X-100 lysis buffer for one hour.Lysates were centrifuged for 15 minutes at 15,000×g. Nucleolin wasprecipitated as described above and eluted with Laemmli sample buffer(1% SDS, 100 mM NH₄HCO₃). Eluted proteins were separated by 8-20%SDS-PAGE and transferred to nitrocellulose. The nitrocellulose membraneswere subsequently incubated, after blocking, with ExtrAvidin-peroxidaseconjugates diluted 1:5,000 (Sigma) for one hour at room temperature.Signals were detected after incubation with ECL reagent and exposure toBiomax MR.

Nucleolin was also detected at the cell surface by FACS analysis withrabbit polyclonal anti-nucleolin antibody prepared against amino acids221 to 232 of nucleolin (NCL3). Cells which were alive and intact asindicated by gating for cells that did not stain with propidium iodidedemonstrated significant binding of the anti-nucleolin antibody (seeFIG. 7B). As shown in the figure, the positive control anti-α5 integrinantibody gave a strong shift, reflecting a high level of cell surfaceexpression of α5β1 integrin. Anti-nucleolin antibody also caused asignificant shift of the FACS peak compared to the control, indicativeof nucleolin cell-surface expression. These results demonstrate thatnucleolin is expressed at the cell surface in MDA-MB-435 cells.

Rabbit polyclonal antibodies were raised against peptides synthesizedaccording to the nucleolin sequence. NCL2, NCL3 and NCL4 were raisedagainst amino acids 43-51, 221-232 and 393-407 of human nucleolin,respectively, and were affinity-purified on the immunizing peptide. Eachpolyclonal antibody immunoblotted the 110-kDa nucleolin band in cellextracts, and the anti-NCL2 and anti-NCL3 antibodies bound intact cellsshown to express cell surface nucleolin.

FACS analysis of cell surface nucleolin was performed as follows.MDA-MB-435 cells (1×10⁶ cells/sample) were detached with EDTA andincubated with primary antibody (10 μg/ml) for 45 minutes on ice. Cellswere washed with ice-cold phosphate-buffered saline (PBS) and incubatedwith Alexa-435 secondary antibody (1:50 in PBS). As a negative control,cells were incubated with secondary antibody only. After washing, cellswere resuspended in 50 μl PBS containing 2 μg/ml propidium iodide todistinguish between living and dead cells. Analysis was performed with10,000 cells per sample using a FACSCalibur flow cytometer (BDBiosciences; San Jose, Calif.).

C. Inhibition of Peptide SEQ ID NO: 9 Internalization by Treatment ofCells with Anti-Nucleolin Antibodies

Nucleolin has been observed to shuttle between the cytoplasm and thenucleus, or the cell surface and the nucleus (Shibata et al., Mol. Cell.Biol. 22:6788-6796 (2002), and Said et al., J. Biol. Chem.277:37492-37502 (2002)). Fluorescein-labeled peptide was used to analyzenucleolin-dependence of SEQ ID NO: 9 localization. For internalizationexperiments, cells were incubated with FITC-labeled peptide (1 μM) fortwo hours at 37° C. After washing with PBS, cells were fixed with 4%paraformaldehyde (PFA) and analyzed by fluorescence microscopy. As shownin FIG. 8A, fluorescein-labeled peptide SEQ ID NO: 9 was taken up byMDA-MB-435 cells and localized to the nucleus. Thus, like other basiccell-penetrating peptides, the HMGN2 peptide SEQ ID NO: 9 is transportedto the nucleus following internalization.

To analyze whether nucleolin is involved in internalization and nucleartransport of peptide SEQ ID NO: 9, cells were co-incubated with ananti-nucleolin antibody prepared against the amino-terminal acidicdomain (NCL3) of nucleolin. As shown in FIG. 8B, co-incubation withanti-NCL3 antibodies abolished cellular uptake and nuclear localizationof labeled peptide SEQ ID NO: 9. An anti-nucleolin antibody prepared byimmunizing against NCL2 (amino acids 43-51), bound to intactnucleolin-expressing cells, but did not inhibit uptake or nuclearlocalization of peptide SEQ ID NO: 9 (see FIG. 8C). Moreover, the NCL3antibody itself was internalized and transported into the nucleus,whereas the NCL2 antibody remained at the cell surface. Internalizationof another peptide that binds MDA-MB-435 cells, FITC-LyP-1 (Laakkonen etal., Nat. Med. 8:751-755 (2002)), was not influenced by anti-nucleolinantibodies (FIGS. 8D and E). These results demonstrate that SEQ ID NO: 9binds to the amino-terminal acidic domain of cell surface-expressednucleolin and that internalization of HMGN2-derived peptides depends onnucleolin, which also may be responsible for nuclear transport. Theseresults further demonstrate that anti-nucleolin antibodies also can beinternalized by cells that express cell surface nucleolin.

D. Internalization of Peptide SEQ ID NO: 9 into MDA-MB-435 Cells isIndependent of Heparin Sulfates

Previous studies have show that binding to heparin sulfates can besufficient for internalization of heparin sulfate-binding proteins(Roghani and Moscatelli, J. Biol. Chem. 267:22156-22162 (1992)). Asdescribed above, peptide SEQ ID NO: 9 is a highly basic molecule,suggesting that negatively charged cell surface glycosaminoglycans suchas heparin sulfate may play a role in binding and internalization ofthis peptide.

Cells that lack glycosaminoglycans including heparin sulfates due to amutation in xylosyl transferase were assayed for the ability to bind andinternalize peptide SEQ ID NO: 9. In particular,glycosaminoglycan-deficient pgsA-745 cells were assayed for the abilityto bind SEQ ID NO: 9-displaying phage as an indication of SEQ ID NO: 9peptide binding. As shown in FIG. 9A, SEQ ID NO:9-displaying phage boundto pgsA-745 cells at about 20% of the binding observed withcorresponding wild type cells. SEQ ID NO: 9-phage binding toglycosaminoglycan-deficient cells was stronger than binding ofnon-recombinant control phage. Furthermore, glycosaminoglycan-deficientpgsA-745 cells incubated with FITC-labeled peptide SEQ ID NO: 9 showedequally efficient uptake and nuclear localization of peptide SEQ ID NO:9 as did wild type cells. Neither cell type internalizedfluorescein-labeled control peptide. In sum, these results indicate thatglycosaminoglycans can contribute to cell surface binding ofHMGN2-derived peptides. However, the demonstration that CHO cellslacking heparin sulfate and other glucosaminoglycans internalize SEQ IDNO: 9 efficiently excludes a direct role for heparin sulfate in cellularuptake of HMGN2-derived peptides such as SEQ ID NO: 9.

Binding of SEQ ID NO: 9-displaying phage to CHO-K1 and pgsA-745 cellswas assayed as follows. CHO-K1 and pgsA-745 cells were grown in alphaMEM Earle's salt with 10% fetal calf serum and 1% Glutamine Pen-Strep(Irvine Scientific; Santa Ana, Calif.). After detaching cells with 2.5mM EDTA, 10⁶ cells were incubated with 10⁸ phage for three hours on ice.Following extensive washing, bound phage were eluted by addition of 100μl BLT5615 bacteria, and titers determined by routine methods.

E. Sub-Cellular Distribution of Nucleolin Changes Depending on GrowthState

To test the hypothesis that localization of nucleolin is affected by thegrowth state of cells cultured in vitro, cells grown under differentconditions were stained with anti-nucleolin antibodies. As shown in FIG.10, anti-NCL3 (anti-nucleolin) antibody stained the surface of activelygrowing MDA-MB-435 cells; there was no surface staining of theMDA-MB-435 cells rendered stationary by serum withdrawal. Nuclearnucleolin was detected in permeabilized cells under both conditions.Thus, nucleolin is exclusively nuclear in serum-starved cells. Theseresults demonstrate that nucleolin is selectively expressed on the cellsurface of proliferating cells and indicate that nucleolin-bindingmolecules can be useful for selectively targeting moieties to activelydividing endothelial and tumor cells.

For nucleolin staining, cells were fixed with 4% paraformaldehyde andeither directly stained with anti-NCL3 antibody (10 μg/ml) or stainedfollowing permeabilization with Triton X-100. Bound antibody wasdetected with Alexa-594 labeled anti-rabbit antibody (Molecular Probes;Eugene, Oreg.) and visualized by fluorescence microscopy.

F. Nucleolin is Expressed in Tumor Vasculature in Vivo

In vivo tissue expression of cell surface nucleolin was analyzed byinjecting the anti-NCL3 antibody intravenously into mice. Tissuescollected 60 minutes after the injection showed selective accumulationof the antibody in tumor blood vessels (FIGS. 11A and B), mimicking thedistribution of peptide SEQ ID NO: 9 shown above. No anti-NCL3 antibodywas detected in the blood vessels of various normal tissues (shown forthe skin in FIG. 11C). A control antibody prepared in the same manner asthe anti-nucleolin antibody did not appear in tumor blood vessels (seeFIG. 11D). These results indicate that nucleolin is selectivelyexpressed on the cell surface of tumor blood vessels in vivo.

In vivo distribution of cell surface nucleolin was examined usingMDA-MB-435 xenografts generated by subcutaneous injection of 10⁶exponentially growing cells in 200 μl culture media. Mice (Balb/c nu/nu;Animal Technologies, Ltd; Fremont, Calif.) were used for furtherexperiments eight weeks after injection. Polyclonal rabbitanti-nucleolin antibody (200 μg) was injected into blood circulation oftumor bearing mice. After one hour of circulation, mice were sacrificedby perfusion of 10 ml PBS into the heart, followed by the injection of4% PFA. Tumor and control tissues were removed and frozen in OCTembedding medium (Tissue-Tek; Elkhart, Ind.). Tissue sections of 5 μmwere used for blood vessel staining with anti-CD31 antibody (Pharmingen;San Diego, Calif.). Injected rabbit antibody and anti-CD31 antibody weredetected with Alexa-594 and Alexa-486 conjugated secondary antibodies,respectively, and examined under an inverted fluorescent microscope.Nuclei were counterstained using 4′6-diamidino-2-phenylindole (Vector;Burlingame, Calif.).

1. An isolated homing peptide comprising the amino acid sequenceKDEPQRRSARLSAKPAPPKPEPKPKKAPAKK (SEQ ID NO: 9) said peptide having alength of less than 70 residues, wherein said peptide specifically bindsnucleolin expressed on the surface of a tumor cell.
 2. The isolatedpeptide of claim 1, which has a length of less than 60 residues.
 3. Theisolated peptide of claim 1, which has a length of less than 50residues.
 4. The isolated peptide of claim 1, which has a length of lessthan 45 residues.
 5. The isolated peptide of claim 1, which has a lengthof less than 40 residues.
 6. The isolated peptide of claim 1, which hasa length of less than 35 residues.
 7. An isolated homing peptide thatselectively homes to tumor blood vessels or tumor cells, comprising theamino acid sequence KDEPQRRSARLSAKPAPPKPEPKPKKAPAKK (SEQ ID NO: 9), saidpeptide having a length of less than 70 residues.
 8. The isolated homingpeptide of claim 7, which has a length of less than 60 residues.
 9. Theisolated homing peptide of claim 7, which has a length of less than 50residues.
 10. The isolated homing peptide of claim 7, which has a lengthof less than 45 residues.
 11. The isolated homing peptide of claim 7,which has a length of less than 40 residues.
 12. The isolated homingpeptide of claim 7, which has a length of less than 35 residues.
 13. Anisolated peptide consisting of the amino acid sequenceKDEPQRRSARLSAKPAPPKPEPKPKKAPAKK (SEQ ID NO: 9).
 14. A chimericpolypeptide comprising a peptide consisting of the amino acid sequenceKDEPQRRSARLSAKPAPPKPEPKPKKAPAKK (SEQ ID NO: 9) fused to a heterologouspolypeptide, wherein said chimeric polypeptide specifically bindsnucleolin.
 15. A chimeric polypeptide comprising a peptide consisting ofthe amino acid sequence KDEPQRRSARLSAKPAPPKPEPKPKKAPAKK (SEQ ID NO: 9)fused to a heterologous polypeptide, wherein said chimeric polypeptideselectively homes to tumor blood vessels or tumor cells.